Flavivirus and alphavirus virus-like particles (vlps)

ABSTRACT

Described herein are flavivirus virus-like particles (VLPs) that display on their surfaces antigenic flavivirus proteins. Also described are methods of making and using these VLPs.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application No.62/184,738, filed Jun. 25, 2015 and U.S. Provisional Application No.62/292,936, filed Feb. 9, 2016, the disclosures of which areincorporated herein by reference.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This work was supported in part by a Qualifying Therapeutic DiscoveryProject Grant form Health and Human Service (HHS) for the Development ofa Multivalent Dengue Virus-Like Particle (VLP) Vaccine to TechnoVax,Inc. The U.S. Government may have certain rights in this invention.

TECHNICAL FIELD

The present invention relates to compositions comprising flavivirus(e.g., dengue or Zika) and/or alphavirus (e.g., chikungunya) virus-likeparticles (VLPs) and to methods of making and using these VLPs,including the creation and production of virus-like particle (VLP) basedvaccines (e.g., for dengue, Zika, and/or chikungunya) as well as its usefor diagnostic and therapeutic indications. In particular, the presentdisclosure includes strategies and methods used for the development ofnovel monovalent or multivalent vaccines that are able to protect humansagainst infection with one or more clades or antigenic variants of theflavivirus (dengue, Zika) and/or alphavirus viruses. Also describedherein are VLP production methods that produce VLPs that display certainantigenic configurations. These VLPs feature conformational epitopesrelevant for the generation of an enhanced neutralizing immune responseto the virus. Single particle monovalent, bivalent, or multivalent(e.g., tetravalent, for example for the 4 dengue serotypes) VLPs areassembled and used to formulate vaccine compositions, which allows forimmunization and subsequent protection against one or more clades orantigenically distinct virus (e.g. Asian clade, South America clade,etc. for Zika; 1, 2, 3, or 4 for dengue serotypes). Furthermore, VLPsare also used for the diagnosis of infection or for therapeuticindications. VLP vaccines can be produced in suspension culture ofeukaryotic cells and released into the culture medium. Afterpurification, concentration, and formulation the vaccine can beadministered by any suitable route, for example, via either mucosal orparenteral routes, and induce an immune response able to protect againstany or all of the Zika, dengue, chikungunya virus clades, antigenicvariants or serotypes. VLPs comprising combinations of Zika, dengueand/or chickungunya and methods of providing immune responses toadditional viruses are also provided.

BACKGROUND

Flaviviruses such as dengue and Zika and alphaviruses such aschikungunya are the causative agents of infections in humans and birthdefects when pregnant women are infected with Zika. Zika fever diseaseresults from an infection with Zika virus (ZIKV), which is transmittedto human by the bite of an infected Aedes mosquito (A. aegypti, A.albopictus and polynesiensis). Zika virus was isolated for the firsttime in the Zika Forest in Uganda from a Rhesus monkey in 1947 and laterfrom humans in 1952. ZIKV belongs to the flavivirus genus within theFlaviviridae family. Members of this family possess a single strandedpositive sense RNA genome (˜10,794 nucleotides long) that encodes onlyone open reading frame (ORF) translated into a single polyprotein whichis cleaved by both cellular and virus-encoded proteases into threestructural proteins (C, prM and E) and seven non-structural proteins(NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5.) that enables virusreplication. Zika virus protein processing and maturation appears to besimilar to that of other members of the family and it occurs through thesecretory pathway beginning with the self-cleavage of NS3 protease withits cofactor NS2B. Then, the NS2B/NS3 complex cleaves the cytoplasmictail of the C protein and host cell signalases and proteases performother cleavages within the polyprotein.

During replication and virus morphogenesis, which occurs in closedassociation with intracellular membranes, nascent virions are assembledand transported through the secretory pathway and released at the cellsurface. Enveloped virions are composed of a cell-derived lipid bilayerencapsulating the C-protein wrapped viral RNA genome and studded withmultiple copies of the proteins E and M. During maturation within thesecretary pathway (trans-Golgi network) the precursor prM protein iscleaved by the host cell's furin protease to produce the small M proteinand the fragment pr, which is released upon virus egress from the cell.The surface of the virus displays E protein (dimers arranged in head totail herringbone arrays) as the major antigenic determinant of the virusand mediates receptor binding and fusion during virus entry into cells.Structural studies of an analogous protein of the genus flavivirusreveals three domains, DI, DII and DIII followed by two helices and twotransmembrane domains, which anchor this protein to the surface of thevirion particle (Crill W D, Chang G-JJ, 2004, Localization andCharacterization of Flavivirus Envelope Glycoprotein Cross-ReactiveEpitopes. J. Virol 78(24):13975-13986). Therefore, this protein is amajor target of the host immune response and a suitable candidate forvaccine development and diagnostic applications.

ZIKV has been transmitted in Africa for many years through a sylvaticcycle between the mosquito vectors and nonhuman primates, withoccasional human infections. In recent years, however, epidemics of Zikahave resulted from cycles of transmission between vectors and humansspreading the disease beyond the African continent into the FrenchPolynesia and other Pacific regions. Since 2015 a dramatic spread ofZIKV that started in Brazil is taking place in South America and theCaribbean Islands and some sporadic cases of travelers have beenidentified in the USA and Europe. Although Zika fever appears to cause amild illness in 1 of 5 people infected, contracting the virus duringpregnancy has been associated with birth defects, primarily microcephaly(defective brain development). Furthermore, an increase of cases ofGuillain-Barre syndrome has been observed following ZIKV infection. Theseriousness of these disorders imposes a tremendous burden on publichealth and human life. In addition to vectors transmission, ZIKV canalso be transmitted by sexual contact, making disease control moredifficult.

It has been observed with other flaviviruses that in addition to matureand immature particles (virions that carry uncleaved pr peptide)produced by flavivirus-infected cells; small non-infectious particlescomposed of M and E are also assembled and released. Furthermore, it wasshown that recombinant expression of proteins prM and E from tick-borneencephalitis (TBE) virus was sufficient to drive assembly and budding ofthis type of particle. Other flavivirus sub-viral particles assembledwith prM and E proteins have also been produced in the yeast Pichiapastoris expression system as well as in mammalian cells. Vaccinecompositions containing these sub-viral particles have been shown toinduce neutralizing antibodies and specific cytotoxic T lymphocyteresponses in mice.

Dengue fever results from infection with dengue virus, which istransmitted to humans by the bite of infected Aedes mosquitoes (A.aegypti, A. albopictus and A. polynesiensis). This mosquitos-borneillness is responsible for 100 million cases of dengue each yearworldwide. The World Health Organization (WHO) estimates that two-thirdof the world human population is at risk of contracting dengueinfection. Furthermore, the relentless spread of the mosquito vectors inrecent years continues to expand the illness to new regions of theworld. Four distinct virus serotypes (DENV1-4) can be transmitted byinfected Aedes mosquitoes causing an infection characterized by fever,headache, myalgia, arthralgia and, depending on the severity of theinfection, may progress to Dengue hemorrhagic fever/Dengue shocksyndrome (DHF/DSS) (WHO (2014) Dengue and severe Dengue Fact Sheet No117. Available at:http://www.who.int/mediacentre/factsheets/fs117/en/[Accessed Sep. 4,2014]). These life-threatening outcomes are more common followingsubsequent infections with a dengue virus of a different serotype. Thepresence of a low concentration of poorly neutralizing antibodiesproduced after the primary infection, appears to heighten infection ofFc-receptor bearing cells, increasing virus replication and clinicalsigns by the mechanism of antibody-dependent enhancement (ADE) ofdisease. This complex interaction between the host-immunity and dengueviruses has hindered the development of a safe and effective denguevaccine, which has to elicit a robust and balanced neutralizing antibodyresponse against each one of the four virus serotypes in order to avoidpotential induction of ADE.

Dengue virus is an enveloped positive sense single-strand RNA virus,which belongs to the flaviviridae family within the Flavivirus genus.The dengue RNA genome (˜10.7-Kb) encodes only one open reading frame(ORF) and translates into a single polyprotein cleaved by both cellularand virus-encoded proteases into three structural proteins (C, prM andE) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4Band NS5.) that enables virus replication (Lindenbach B D, Rice C M,2003, Molecular biology of flaviviruses. Adv Virus Res 59:23-61).Protein processing and maturation occur through the secretory pathwayand begins with the self-cleavage of NS3 protease with its cofactorNS2b. Then, the NS2B/NS3 complex cleaves the cytoplasmic tail of the Cprotein and host cell signalases and proteases perform other cleavageswithin the polyprotein.

During replication and virus morphogenesis, which occurs in closelyassociation with intracellular membranes, nascent virions are assembledand transported through the secretory pathway and released at the cellsurface. Enveloped virions are composed of a cell-derived lipid bilayerencapsulating the C-protein wrapped viral RNA genome and studded withmultiple copies of the proteins E and M. During maturation within thesecretary pathway (trans-Golgi network) the precursor prM protein iscleaved by the host furin protease to produce the small M protein andthe fragment pr, which is released upon virus egress from the cell. Thesurface of the virus displays E protein (dimers ordered in head to tailherringbone arrays) as the major antigenic determinant of the virus andmediates receptor binding and fusion during virus entry. This proteinhas three domains, DI, DII and DIII followed by two helices and twotransmembrane domains, which anchor this protein to the surface of thevirion particle (Crill W D, Chang G-JJ, 2004, Localization andCharacterization of Flavivirus Envelope Glycoprotein Cross-ReactiveEpitopes. J Virol 78(24):13975-13986). Therefore, this protein is amajor target for vaccine development. It has been observed that inaddition to mature and immature particles (virions that carry uncleavedpr peptide) produced by dengue-infected cells; small non-infectiousparticles composed of M and E are also assembled and released.Furthermore, it was shown that recombinant expression of proteins prMand E from tick-borne encephalitis (TBE) virus was sufficient to driveassembly and budding of this type of particle. Dengue sub-viralparticles assembled with prM and E proteins have also been produced inthe yeast Pichia pastoris expression system as well as in mammaliancells. Vaccine compositions containing these sub-viral particles havebeen shown to induce neutralizing antibodies and specific CTL responsesin mice.

At this time there is no vaccine or specific treatment to control,combat or prevent ZIKV infection. For their parts, dengue vaccines arebeing developed using conventional strategies such as chimeric,live-attenuated, and inactivated viruses or DNA and some of thesevaccine are far advanced in their development. However, due to a prolongimmunization regimen, as well as unbalanced and insufficient immunityagainst some serotypes raises concern as to whether these vaccines aresafe and efficacious. Therefore, new technologies are needed to developsafer and more effective dengue vaccines. The prevention of infection byvaccination represents a critical unmet need to control the spread andthe effects of theses diseases globally. Here, we described theformation of virus-like particles as a strategy for flavivirus (Zika anddengue) and/or alphavirus (chickungunya) vaccine development andformulations for an specific virus as example dengue containing foursertoypes or Zika containing one or more antigenic variants as well ascombination as dengue, Zika and chikungunya and alternative dualcompositions e.g. dengue/Zika or dengue/Chikungunya or Zika/Chikungunya.Furthermore, the VLPs as described herein can be used for diagnostic aswell as therapeutic applications.

SUMMARY

Described herein are virus-like particles (VLPs) comprising at least oneantigenic flavivirus or alphavirus protein (Zika, dengue orchickungunya). Also described are compositions comprising these VLPs, aswell as methods for making and using these VLPs. The VLPs describedherein are devoid of viral genetic material and therefore unable toreplicate or cause infection; however given their morphological,biochemical and antigenic similarities to wild type virions, VLPs arehighly immunogenic and able to elicit robust protective immuneresponses. Unlike virion inactivated based vaccines, VLPs are notinfectious eliminating the need for chemical treatment, thus maintainingthe native conformation of structural components and antigenic epitopes.

Thus, the invention describes a novel approach for Zika, dengue and/orchickungunya virus-like particle (VLP) development. In particular, wedescribe the creation, development and production of VLP vaccines forZika, dengue as well as chickungunya that will trigger, upon humanimmunization, a strong and balanced immune response characterized by theinduction of high level of neutralizing antibodies. In certainembodiments, the VLP triggers a high level of neutralizing antibodiesagainst the four-dengue virus serotypes and/or multiple Zika cladesconcurrently. In other embodiments, the VLP vaccine as a virus specificcomposition triggers a high level of neutralizing antibodies againsteither the four-Dengue serotypes, or against a single or multiple Zikaclades. In another embodiment, a combination vaccine elicits a highlevel of neutralizing response against the four-dengue serotypes, Zikaclades and chikungunya.

Based on flavivirus subviral particles studies as well as on our ownexperience in virus-like particle assembly, we have designed a new andmore effective strategy for the formation and release of virus-likeparticles (e.g., dengue and/or Zika). We have found that theco-expression of flavivirus (dengue, Zika) virus structural proteinscapsid (C), preMembrane (prM), envelope (E) together with thenon-structural protein NS2B/NS3 drives the assembly and release ofvirus-like particles. The presence of the complex NS2B/NS3 contributesnot only to the processing of the polyprotein CprME by its proteasefunctions but also to the particles assembly and release. We alsoproduce VLPs displaying E protein with different reactivites asdemonstrated with a monoclonal antibody that recognizes a conformationepitope of the E protein that is shared by other flaviviruses. Thesedifferent E protein conformations seem to be highly relevant for theelicitation of potent neutralizing antibody in humans.

In one aspect, described herein is a flavivirus (e.g., dengue, Zika,yellow fever, Japanese encephalitis, tick-borne encephalitis, hepatitisC and/or West Nile virus) virus-like particle (VLP) comprising at leastone flavivirus structural protein and at least one non-structuralflavivirus protein. In certain embodiments, the VLP comprises all of theCPrME proteins. Any CPrME proteins can be employed, including wild-typeor mutated (e.g., codon optimized) sequences from any flavivirus speciesand serotype (dengue 1, 2, 3, 4, Zika, etc.). In an exemplaryembodiment, the wild-type nucleotide sequence of CprME of dengue-2 isshown in SEQ ID NO:1, and the amino acid sequence is described in SEQ IDNO:2. Other wild-type CprME sequences are known in the art and may bereadily aligned with any of the exemplary Zika or dengue (e.g.,dengue-2) sequences disclosed herein. In certain embodiments, the CprMEsequence comprises a sequence with one or more mutations (substitutions,additions and/or deletions) as compared to wild-type and/or a codonoptimized sequences. See, e.g., SEQ ID NO:3 (mutated) and SEQ ID NO:4(codon optimized with mutations), which both include the amino acidsequences shown in SEQ ID NO:5. In other embodiments, the VLP consistsof less than all of the CPrME proteins (e.g., CPrME, PrME, CME, CPrE orME), as compared to the full length wild-type or mutated sequences.Similar mutations can be made in any flavivirus CprME protein followingthe teachings described herein. In any of the assembly of VLPs asdescribed herein the non-structural proteins may comprise NS2B and/orNS3 proteins derived from any flavivirus species or serotype (e.g. anexample of wild type nucleotide sequence of NS2B/NS3 is shown in SEQ IDNO:6 and amino acid sequence described in SEQ ID NO:7) or modified(e.g., truncated, mutated and/or codon optimized) proteins (e.g. exampleof modified NS2B/NS3 nucleotide sequence is shown in SEQ ID NO:8 andamino acid sequence shown in SEQ ID NO:9). NS2B or NS3 may also be usedas single proteins the nucleotide sequences (e.g., fragments derivedfrom any flavivirus NS2B/NS3 protein. Exemplary, non-limiting sequencesare shown in SEQ ID NO: 10 (NS2B nucleotide sequence), SEQ ID NO:11(NS2B amino acid sequence), SEQ ID NO:12 (NS3 nucleotide sequence) andSEQ ID NO: 13 (NS3 amino acid). The VLP may be monovalent, bivalent ormultiple valent and display on its surface one or more antigenicflavivirus proteins (1, 2, 3, 4 or more proteins): from a singleflavivirus, from one or more serotypes (or clades or isolated) of asingle flavivirus (e.g., a bivalent or multivalent; from multipleflaviviruses (e.g., dengue and/or Zika and/or alphaviruses), as well ascombinations thereof.

Also provided is an immunogenic composition comprising at least one VLPas described herein. In certain embodiments, the immunogeniccompositions further comprise an adjuvant. Thus, described herein areflavivirus (e.g., dengue, Zika) virus-like particles (VLP)—also known assubviral particles, recombinant subviral particles, biologicalnanoparticles, nanoparticles, etc.—utilizing structural (C-prM-E) andnon-structural (NS2B/NS3) viral proteins. These VLPs are designed asvaccine or immunogens for protecting against infection with any one ofthe four-dengue virus serotypes and/or any of the known Zikaclades/isolates. In certain embodiments, the VLP also comprisesalphavirus antigenic proteins (e.g., chickungunya).

Also provided are DNA constructs comprising sequences encodingflavivirus viral proteins (structural and non-structural) used toassemble the VLP of any of claims 1 to 8. The constructs may furthercomprise one or more sequences encoding one or more linkers between oneor more of the sequences encoding the structural and non-structuralproteins (e.g., a linker comprising amino acids corresponding to aminoacids 1 to 8 or 9 or 10 of any flavivirus NS1 protein, numbered relativeto any flavivirus NS1 protein, for example as shown in SEQ ID NO:15(amino acid) (SEQ ID NO:14 shows the nucleotide sequence encoding theseresidues); a linker comprising amino acids corresponding to 186 or 187or 188 or 189 to amino acids corresponding to 218 or 225 of anyflavivirus NS2A protein, numbered relative to any wild-type protein(e.g. as shown in SEQ ID NO:17); a linker comprising amino acid 1 to 8or 9 or 10 of NS1, amino acids 1 to 24 or 25 or 26 or 27 or 28 or 29 or30 or 31 or 32 of NS2A, amino acids 186 or 187 or 188 or 189 to 218 or225 of NS2A; a linker comprising amino acids 1 to 8 or 9 or 10 of NS1and the second transmembrane domain of NS2B (e.g. nucleotide sequenceamino acid sequence SEQ ID NO:11); a linker comprising amino acid 1 to 8or 9 or 10 of NS1 and the first transmembrane domain of NS2A (e.g.,amino acids encoded by nucleotides 51 to 100 of nucleotide sequence, SEQID NO: 16 and amino acid sequence SEQ ID NO: 17); and a linkercomprising amino acid 1 to 8 or 9 or 10 of NS1 and the C terminalportion of NS2B comprising the second transmembrane domain to the end ofthe protein. The DNA constructs may comprise the flavivirusprotein-encoding sequences in any order (e.g., a full length NS2B (e.g.SEQ ID NO:10): and a full NS3 (e.g. SEQ ID NO: 18) or modified truncatedNS2B/NS3 (SEQ ID NO: 8) or wild-type NS2B/NS3 (e.g. SEQ ID NO: 6)operably linked directly to the structural proteins CprME (e.g. SEQ IDNO: 4) in any order). In certain embodiments, the furin proteasecleavage site between pr and M protein of the constructs describesherein (e.g., SEQ ID NO:5) is modified by substituting amino acidsresidues at position P3 with hydrophobic one and/or wherein the NS3protease active site is modified (e.g. as shown in NS3 alone SEQ ID NO:13 and NS2B/NS3 SEQ ID NO: 9, which correspond to nucleotide sequencesSEQ ID NO: 12 and SEQ ID NO: 8) in such that its enzymatic activity isenhanced. In other embodiments, sequence(s) encoding the E protein (e.g.SEQ ID NO: 47) is (are) modified to enhance VLP assemble and release(e.g., amphipathic helix 1 in the stem domain of the E protein ismodified to enhance the hydrophobic properties of one side of the helix;one, two, three or more amino acids in the hydrophobic side of the helixare substituted, for example, at positions corresponding to 398, 401and/or 412 (e.g., numbered relative to SEQ ID NO:47), including but notlimited to I398L, I398M, I398V, I398A or M401A, M401L, M401V, M401I, orM412A, M412L, M412V, M412I; and/or helix 1 and/or helix 2 of the Eprotein (e.g. as example I398L, M401A, and M412L of SEQ ID NO: 19) areexchanged with the helix sequences of other flaviviruses or analogousmotif from other viruses and cellular sources).

In another aspect, methods of generating (assembling) the VLPs describedherein are provided. In certain embodiments, such methods and strategiesinvolve mutations, deletions, insertions, gene organization and/orexpression conditions to enhance particle morphogenesis and egress fromproducing cells. Also delineated are strategies for the assembly of VLPsdisplaying on its surface the E protein of a single serotype(monovalent), or the E protein of two distinct serotypes (bivalent), orthe E protein of three distinct serotypes (trivalent) or the E proteinof each one of the four-dengue virus serotypes (tetravalent) or againmultiple Zika clades. In certain embodiments, combining VLPs withalternative antigenic composition allows for the formulation of atetravalent vaccine. Furthermore, production of VLP can be attained insuspension cultures of transfected eukaryotic cells following theexpression of the selected structural and non-structural genes.Transient or stable transfection methods can be used to introduce intocells the plasmids that direct proteins expression. VLPs are releasedfrom the producing cells into the culture medium from where they arecollected and purified by different methods such as gradientcentrifugation, filtration and chromatography or combination thereof.Thus, also provided is a method of producing a VLP, the methodcomprising introducing into a host cell (e.g., a eukaryotic cells suchas a mammalian, yeast, insect, plant, amphibian and avian cells) one ormore DNA constructs as described herein under conditions such that thecells produces the VLP. In certain embodiments, host cell(s) arecultured at temperatures ranging from 25° C. to 33° C. (e.g., 25° C.,26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C. or 33° C.). Alsoprovided are VLPs generated by the methods as described herein as wellas a method of generating an immune response to one or more flavivirusesin a subject (e.g., human), the method comprising administering (e.g.,mucosally, intradermally, subcutaneously, intramuscularly, or orally) tothe subject an effective amount of the VLPs and/or immunogeniccompositions as described herein. The methods described herein canresult in an immune response that treats and/or prevents (vaccinates)the subject against multiple serotypes or clades of one or moreflaviviruses.

In yet another aspect, described herein is the formation of VLPscontaining E proteins of different structural conformations resultingfrom the production at different temperatures (e.g. 37° C. or 31° C.).These VLPs show differential reactivity with a specific monoclonalantibody that recognizes the E protein, reflecting their conformationaldifferences. In addition, VLPs produced at 31° C. elicited strongertiters of neutralizing antibodies than those induced by VLP produced at37° C. when administered as vaccine to small animal models. Atetravalent VLP based vaccine can be formulated with single tetravalentVLPs (one particle carrying E antigens of all serotypes/clades) or byblending VLP of alternative antigenic compositions. The utility of theVLPs as described herein may include, but it is not limited to, vaccineand immunological use, as adjuvant, and/or immune-modulators, deliveryvehicle for heterologous proteins or small molecules and RNA moleculesas well as prophylactic and therapeutic applications.

In another aspect of the invention described provides dengue and/or Zikavirus-like particles (VLP) (also known as subviral particles),recombinant subviral particles, biological nanoparticles, nanoparticles,etc.—utilizing structural prM-E or C-prM-E and non-structural (NS2B/NS3)viral proteins. These VLPs are designed as vaccine or immunogens forprotecting against infection with any one of the dengue and/or Zikavirus clades, antigenic variants or serotypes. Furthermore, theflavivirus (e.g., dengue/Zika) vaccine could be combined with one or allof other VLP based vaccine such as dengue, yellow fever, West Nile, orchikungunya, which are viral diseases transmitted by mosquito vectors.

In addition, methods for generating (assembling) the VLPs (e.g., dengueand/or Zika) described herein are provided. In certain embodiments, suchmethods and strategies involve mutations, deletions, insertions, geneorganization arrangements and the conditions under which the VLPs areproduced recombinantly to better retain particle resemblance to thevirus and enhance egress from the producing cells. Also delineated arestrategies for the assembly of VLPs displaying on their surface the Eprotein of a single clade/antigenic variant or the E protein of twodistinct clades/antigenic variants (bivalent), or the E protein ofdistinct clades/antigenic variants (multivalent). In certainembodiments, combining VLPs with alternative antigenic compositionallows for the formulation of a multivalent vaccine. Furthermore,production of VLPs can be attained in suspension cultures of eukaryoticcells following the expression of the selected structural proteins prMEor CprME alone or structural and non-structural proteins NS2B/NS3combined. Transient or stable transfection methods can be used tointroduce into cells the plasmids that direct proteins expression. VLPsare released from the producing cells into the culture medium from wherethey are collected and purified by different methods such as gradientcentrifugation, filtration and chromatography or combination thereof.

In yet another aspect, described herein is the formation of VLPscontaining E proteins of different structural conformation resultingfrom the production of the VLPs at different temperatures, one setranging from 27° C.-to 33° C. (e.g. 31° C.) and a second set rangingfrom 34° C. to 41° C. (e.g. 37° C.). These VLPs show differentialreactivity with a specific monoclonal antibody that recognizes sharedepitopes of E protein amongst other flaviviruses and reflects theirconformational differences when produced at distinct temperatures. Inaddition, VLPs produced at 31° C. elicits stronger titers ofneutralizing antibodies than those induced by VLP produced at 37° C.when administered as vaccine to small animals. Single or multivalentantigenic VLP based vaccine (Zika, dengue and/or chickunguna) can beformulated (one particle carrying E antigens of several clades or byblending VLP of alternative antigenic compositions). The utility of thethese VLPs may include, but it is not limited to, vaccine andimmunological use, as adjuvant, and/or immune-modulators, deliveryvehicle for heterologous proteins or small molecules as well asprophylactic, therapeutic and diagnostic applications.

Also provided are VLPs produced by any of the methods described herein.

In a still further aspect, provided herein is a method of generating animmune response to a flavivirus and/or alphavirus in a subject, themethod comprising administering to the subject (e.g., human) aneffective amount of a VLP and/or immunogenic composition as describedherein to the subject. In certain embodiments, the composition isadministered mucosally, intradermally, subcutaneously, intramuscularly,or orally. In certain embodiments, the methods generate an immuneresponse to multiple strains or subtypes of flaviviruses, therebyproviding a “universal” vaccine that protects the subject againstinfection from various flaviviruses and/or over time (more than oneseason).

Any of the methods may involve multiple administrations (e.g., amultiple dose schedule).

In another aspect, a packaging cell line is provided for producing VLPsas described herein. The cell line may be stably transfected with one ormore polynucleotides encoding structural proteins and upon introductionand expression of the one or more flavivirus protein-encoding sequencesnot stably transfected into the cell, the VLP is produced by the cell.The packaging cell may be an insect, plant, mammalian, bacterial orfungal cell. In certain embodiments, the packaging cell is a mammalian(e.g., human) cell line.

Thus, the invention includes but is not limited to the followingembodiments:

1. A flavivirus virus-like particle (VLP) comprising the proteins CPrMEthat are assembled following the co-expression of structural andnon-structural proteins, wherein said flavivirus is dengue and/or Zika.

2. A flavivirus virus-like particle (VLP) comprising of the structuralproteins CPrME that are assembled following the expression of the samestructural proteins, wherein said flavivirus is dengue and/or Zika.

3. A flavivirus virus-like particle comprising less than the structuralprotein CPrME such as PrME or CME or CPrE or ME that are assembledfollowing their expression or co-expressed with the non-structuralproteins, wherein said flavivirus is dengue and/or Zika.

4. A virus-like particle of 1, 2 and/or 3, wherein the structuralproteins are produced from separate transcription units.

5. A virus-like particle (VLP) of 1, 2, 3 and/or 4 where thenon-structural proteins comprise the full length or truncated form ofNS3 co-expressed with the full length or truncated forms of NS2B.

6. A DNA construct comprising sequences encoding dengue and/or Zikaviral proteins used to assemble VLPs, wherein the structural andnon-structural viral proteins are operably linked to form a singlesegment with a defined order optionally comprising a linker such as asequence of different portions of the NS1, NS2A and/or NS2B proteins.

7. The DNA construct of 6, wherein the linker comprises amino acidscorresponding to amino acids 1 to 8 or 9 or 10 of NS1 connected to aportion of NS2A comprising of amino acids corresponding to 186 or 187 or188 or 189 to amino acids corresponding to 218 of NS2A.

8. The DNA construct of 6, wherein the linker comprises amino acid 1 to8 or 9 or 10 of NS1 are connected to a first portion of NS2A comprisingamino acid 1 to 24 or 25 or 26 or 27 or 28 or 29 or 30 or 31 or 32connected to a second portion of NS2A comprising of amino acid 186 or187 or 188 or 189 to 218 of NS2A.

9. The DNA construct of 6, wherein the linker comprises amino acid 1 to8 or 9 or 10 of NS1 connected to the second transmembrane domain ofNS2B.

10. The DNA construct of 6, wherein the linker comprises amino acid 1 to8 or 9 or 10 of NS1 connected to the first transmembrane domain of NS2A.

11. The DNA construct of 6, wherein the linker comprises amino acid 1 to8 or 9 or 10 of NS1 connected to the C terminal portion of NS2Bcomprising the second transmembrane domain to the end of the protein.

12. The DNA construct of any of 6 through 11, wherein the order of thestructural and non-structural segments is inverted and thenon-structural segment is operably linked to the structural segment withor without a connecting linker.

13. The DNA construct of 12, wherein the non-structural proteinoptionally includes a full length NS2B and a full NS3 genetically linkeddirectly to the structural proteins CprME.

14. The DNA construct of 12 and 13, wherein the non-structural proteinscomprise a truncated variant of NS3 in which the helicase domain isdeleted but the protease domain and its recognizable self-cleavage sitelocated at the carboxyl terminal of the protein is preserved. Theself-cleavage site serves as one example of a linker that may be used toconnect the non-structural and structural proteins.

15. A DNA construct comprising sequences encoding dengue and/or Zikaviral proteins are used to assemble VLPs wherein the furin proteasecleavage site between pr and M protein is modified by substituting aminoacids residues at position P3 with hydrophobic amino acids.

16. A DNA construct comprising sequences encoding dengue and/or Zikaviral proteins are used to assemble VLPs wherein the helices of the Eprotein are modified to enhance VLP assemble and release.

17. The DNA construct of 16, where the amphipathic helix 1 in the stemdomain of the E protein is modified to enhance the hydrophobicproperties of one side of the helix.

18. The DNA construct of 16 and/or 17 where one, two, three or moreamino acids in the hydrophobic side of the helix are substituted, forexample at positions corresponding to 398, 401 and/or 412, including butnot limited to I398L, I398M, I398V, I398A or M401A, M401L, M401V, M401I,or M412A, M412L, M412V, M412I.

19. A DNA construct of 16, wherein the helix 1 and/or helix 2 areexchanged with the helix sequences of other flaviviruses or analogousmotif from other viruses and cellular sources.

20. A DNA construct comprising sequences encoding flavivirus (Zika,dengue, yellow fever, Japanese encephalitis, West Nile virus etc.) viralproteins used to assemble VLPs wherein the NS3 protease active site ismodified in order to enhance its enzymatic activity. Such modificationmay include but are not limited to the substitution of the amino acidcorresponding to leucine at position 115 to a preferred amino acid witha shorter side chain such as alanine.

21. A method of increasing the amount of mature particles produced by acell, the method comprising enhancing cleavage between pr and M by thefurin protease wherein said protease is furnished to the culture mediaof VLP producing cells, or co expressed with the VLP producing genes orproduced constitutively in stably transfected cells used for VLPproduction.

22. A method of producing VLPs comprising selected gene products (e.g.,dengue and/or Zika proteins), the method comprising transientlytransfecting a eukaryotic cell with one or more plasmids comprisingsequences encoding the selected gene products such that the VLPs areproduced by the eukaryotic cell.

23. A method of producing VLPs comprising selected gene products, themethod comprising stably integrating with one or more sequences encodingthe selected gene products into the genome of a eukaryotic cell suchthat the VLPs are produced by the eukaryotic cell.

24. The method of 22 and 23, wherein said eukaryotic cell is selectedfrom the group consisting of mammalian, yeast, insect, plant, amphibianand avian cells.

25. A method of producing VLPs with selected gene products and distinctstructural conformation of the E, the method comprising transiently orstably transfecting a eukaryotic cell one or more sequences encoding theselected gene products and culturing cells at temperatures ranging from25° C. to 33° C., in which the optimal temperature is 31° C. such thatthe VLPs are produced by the eukaryotic cell.

26. The method of 25, wherein said VLP elicits higher neutralizingantibodies titers in humans or animals and are more protective againstone or all dengue virus serotypes than those induced by VLPs produced athigher temperature (e.g. 37° C.) when administered to said humans oranimals.

27. The method of any of 22 to 26, wherein the VLP is a single bivalentVLP that displays on its surface the E antigen of two dengue virusserotypes (e.g. 1 and 2 or 1 and 3 or 1 and 4 or combination thereof) ora single tetravalent VLP wherein said VLP displays on its surface the Eantigen of all four-dengue virus serotype (e.g. 1, 2, 3 and 4) or asingle bivalent VLP that displays on its surface the E antigen of twoZika virus clades or a single multivalent VLP wherein said VLP displayson its surface the E antigen of multiple antigenic variants/clades ofthe Zika virus.

28. A VLP generated by the method of any of 22 to 27.

29. A single bivalent VLP that displays on its surface the E antigen oftwo dengue virus serotypes (e.g. 1 and 2 or 1 and 3 or 1 and 4 orcombination thereof).

30. A single tetravalent VLP wherein said VLP displays on its surfacethe E antigen of all four-dengue virus serotype (e.g. 1, 2, 3 and 4).

31. A monovalent vaccine comprising at least one of the four-dengueserotypes monovalent.

32. A tetravalent vaccine composed of four monovalent VLPs or twobivalent VLPs wherein said induces strong and balance immune response toall dengue virus serotypes.

33. A single bivalent VLP that displays on its surface the E antigen oftwo or more flavivirus (Zika, dengue and/or yellow fever, Japaneseencephalitis, West Nile virus etc.) virus clades/antigenic variants orcombinations thereof.

34. A single multivalent VLP wherein said VLP displays on its surfacethe E antigen of multiple flavivirus (Zika, dengue and/or yellow fever,Japanese encephalitis, West Nile virus etc.) virus clades/antigenicvariants or serotypes.

35. A monovalent VLP vaccine comprising at least one of the clades offlavivirus virus (monovalent).

36. A multivalent vaccine composed of various monovalent VLPs or twobivalent VLPs wherein said induces strong and balance immune response to(i) one or all virus clades/antigenic variants or serotypes of aflavivirus (e.g., Zika) and/or (ii) one or more virus clades/antigenicvariants or serotypes of at least one other flavivirus (e.g., dengueand/or yellow fever, Japanese encephalitis, West Nile virus) orAlphavirus chikungunya.

37. The VLP or vaccines of any of 31 to 35, wherein the vaccine alsocomprises adjuvant.

38. Flavivirus vaccine compositions where the Zika vaccine is blended inbivalent, trivalent, tetravalent or pentavalent formulations andcombination thereof with VLPs derived from dengue virus, yellow fevervirus, West Nile virus, Japanese encephalitis virus or Alphaviruschikungunya.

39. The VLP or vaccines of any of 29 to 38, wherein the vaccinecomprises an adjuvant. In another aspect, described herein is a hostcell comprising any of the VLPs as described above. In certainembodiments, the host cell permits assembly and release of a VLP asdescribed herein from one or more vectors encoding the polypeptides ofthe VLP. In certain embodiments, the eukaryotic cell is selected fromthe group consisting of a yeast cell, an insect cell, an amphibian cell,an avian cell, a plant cell or a mammalian cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic depicting the structural and non-structural genesselected for virus-like particle assembly and their differentconfigurations.

FIGS. 2A through 2C are schematics depicting exemplary arrangements forconstructs comprising the genes encoding the structural andnon-structural protein utilized for the assembly of virus-likeparticles.

FIGS. 3A through 3E show exemplary antigenic composition of single VLPsand options for formulating multivalent (e.g., tetravalent) vaccines.

FIGS. 4A and 4B are schematics depicting dengue virus genome and VLPassembly strategy. FIG. 4A is a schematic showing the dengue virusgenome, which contains a single open reading frame (ORF) that expressesa polyprotein comprised of both structural and non-structural proteins,which arise via several proteolytic cleavages (top panel). In earlystages, the complex viral protease NS3 with its cofactor NS2B (⬇)self-cleaves before cleaving the capsid protein. A host cell signalaseis also involved in the maturation of the polyprotein (∇). In a finalstep, the furin protease (∇) cleaves the pr portion from the M proteinto uncover E protein fusion peptide. FIG. 4B depicts the VLP assemblystrategy relies on the native properties of the structural genesalthough key point mutations were introduced to improve proteinprocessing and particle assembly. The furin cleavage site (∇) wasmutated at E88A to enhance cleavage at this location. The E protein wasmutated as followed I398A, M401A, and M412L to improve the amphipathicproperties of the helical domain 1 and enhance trafficking and secretionof E. The viral NS3 protein was truncated maintaining only itsN-terminal protease domain that was mutated at L115A to enhance itscatalytic activity. The protease domain is kept as a singletranscription unit together with its cofactor NS2B.

FIGS. 5A through 5D show dengue VLPs were produced in Expi293™ cells byco-expression of CprME-NS2B/NS3 or CprME alone. Analysis of transfectedcells lysates show that capsid protein cleavage was efficient when CprMEprotease was co-expressed with CprME. The distinct cell lysates (20 μgof total protein per lane) were tested by Western blot using specificantibodies: anti-E (shown in FIG. 5A), anti-pr (shown in FIG. 5B),anti-C (shown in FIG. 5C) and anti-NS2B (shown in FIG. 5D) antibodies.

FIG. 6 shows dengue VLP secretion is enhanced by co-expression of CprMEand the viral protease complex NS2B/NS3. Expi293™ cells were transfectedwith CprME alone or CprME together with NS2B/NS3. Cell supernatant frommock transfected or CprME or CprME-NS2B/NS3 transfected cells werepurified by ultracentrifugation. Purified VLPs were analyzed via Westernblot using 20 g of total protein per lane. Purified DENV-2 virus wasused as control (20 μg). The Western blot membranes were probed with(top panel) an anti-E antibody and (bottom panel) an anti-prM antibody.

FIGS. 7A through 7C show analysis of the effect of temperature on thereactivity of the envelope (E) protein display on the surface of thedengue VLP with monoclonal antibodies recognizing conformationalepitopes. Samples of gradient purified VLP fractions were applied tonitrocellulose membranes and probed by dot blot with the followingantibodies: FIG. 7A shows anti-E polyclonal antibody; FIG. 7B shows 4G2MAb; and FIG. 7C shows 3H5 MAb. VLPs produced at a lower temperature(31° C.) demonstrate better protein folding of conformational epitopesas shown by reactivity with MAbs 4G2 and 3H3, which also react withDENV-2 virus control.

FIGS. 8A and 8B show electron microscopy study of gradient purifiedDENV-2 VLP (bar represents 100 nm). FIG. 8A shows negative staining with2% Uranyl acetate. Arrows point to the dengue VLPs. FIG. 8B shows twoparticles observed after Immuno-gold staining with 3H5 monoclonalantibody

FIG. 9 is a graph showing results of ELISA assays at the indicatedconditions. Analysis of anti-DENV specific antibodies (total IgG) inmouse serum (n=4) following immunization with dengue VLP vaccine(TVX-31° C. and TVX37° C.) and inactivated dengue-2 virus control(DENV-2) via chemiluminescent ELISA. Pre-immune sera (Pre I.) showstatistical difference (*) (P<0.05) with the immunized groups.

FIG. 10 is a table showing neutralization power of vaccinated mice serais presented as the reciprocal of serum dilution for which 50% of thevirus is neutralized (PRNT₅₀). PRNT₅₀ is calculated using PROBIT method(Finney, 1952).

FIGS. 11A through 11F shows electron microscopy studies of Zika VLPs.Zika virus-like particles (zVLPs) were purified by ultracentrifugationthrough a potassium tartrate (10%-35%)/glycerol (7%-28%) linear gradientand examined by electron microscopy. FIGS. 11A, 11B and 11C show imagesof negatively stained zVLPs, which have round shape of 60 nm diameter.FIGS. 11D, 11E and 11F show images of immuno-gold labeled zVLP using ananti-E specific MAb as primary and a gold labeled (10 nm beads)secondary Ab. Black dots (beads) demonstrates the presence of the Eprotein on the surface of the particles.

FIG. 12 shows dot blot evaluation of gradient purification profile ofZika virus-like particles (zVLPs) and Zika virus. Aliquots (3 ul) fromeach fraction of gradient purified VLPs and ZIKA virus were tested bydot blot with an anti-E specific MAb. VLPs were detected in fractions 15to 20 whereas the ZIKA virus was detected in fractions 13 to 15.

FIG. 13 shows Western blot examination of gradient purified VLPs andZika virus. The Western blot membrane was probed with an anti-E specificantibody and each lane correspond to the following: (1) Dengue control,2) mock, (3) Zika virus (ZIKV) fractions 6-9, (4) Zika VLPs fractions10-12, and (5) Zika VLPs fractions 7-8. The anti-E antibody detected theE protein of VLPs (lanes 4 and 5) and ZIKV (lane 3) as well as denguecontrol (lane 1).

DETAILED DESCRIPTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, immunology and pharmacology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack PublishingCompany, 1990); Methods In Enzymology (S. Colowick and N. Kaplan, eds.,Academic Press, Inc.); and Handbook of Experimental Immunology, Vols.I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell ScientificPublications); Sambrook, et al., Molecular Cloning: A Laboratory Manual(2nd Edition, 1989); Short Protocols in Molecular Biology, 4th ed.(Ausubel et al. eds., 1999, John Wiley & Sons); Molecular BiologyTechniques: An Intensive Laboratory Course, (Ream et al., eds., 1998,Academic Press); PCR (Introduction to Biotechniques Series), 2nd ed.(Newton & Graham eds., 1997, Springer Verlag); Fundamental Virology,Second Edition (Fields & Knipe eds., 1991, Raven Press, New York).

All publications, patents and patent applications cited herein arehereby incorporated by reference in their entirety.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise. Thus, for example, reference to “a VLP”includes a mixture of two or more such VLPs.

Definitions

As used herein, the terms “sub-viral particle” “virus-like particle”,“recombinant subviral particles” or “VLP” refer to a nonreplicating,viral shell. VLPs are generally composed of one or more viral proteins,such as, but not limited to those proteins referred to as capsid, coat,shell, surface and/or envelope proteins, or particle-formingpolypeptides derived from these proteins. VLPs can form spontaneouslyupon recombinant expression of the protein in an appropriate expressionsystem. Methods for producing particular VLPs are known in the art anddiscussed more fully below. The presence of VLPs following recombinantexpression of viral proteins can be detected using conventionaltechniques known in the art, such as by electron microscopy, biophysicaland immunological characterizations, and the like. See, e.g., Baker etal., Biophys. J. (1991) 60:1445-1456; Hagensee et al., J. Virol. (1994)68:4503-4505. For example, VLPs can be isolated by density gradientcentrifugation and/or identified by characteristic density banding.Alternatively, cryoelectron microscopy can be performed on vitrifiedaqueous samples of the VLP preparation in question, and images recordedunder appropriate exposure conditions. Additional methods of VLPpurification include but are not limited to chromatographic techniquessuch as affinity, ion exchange, size exclusion, and reverse phaseprocedures.

By “particle-forming polypeptide” derived from a particular viralprotein is meant a full-length or near full-length viral protein, aswell as a fragment thereof, or a viral protein with internal deletions,which has the ability to form VLPs under conditions that favor VLPformation. Accordingly, the polypeptide may comprise the full-lengthsequence, fragments, truncated and partial sequences, as well as analogsand precursor forms of the reference molecule. The term thereforeintends deletions, additions and substitutions to the sequence, so longas the polypeptide retains the ability to form a VLP. Thus, the termincludes natural variations of the specified polypeptide sincevariations in coat proteins often occur between viral isolates. The termalso includes deletions, additions and substitutions that do notnaturally occur in the reference protein, so long as the protein retainsthe ability to form a VLP. Preferred substitutions are those which areconservative in nature, i.e., those substitutions that take place withina family of amino acids that are related in their side chains.Specifically, amino acids are generally divided into four families: (1)acidic—aspartate and glutamate; (2) basic—lysine, arginine, histidine;(3) non-polar—alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar—glycine,asparagine, glutamine, cysteine, serine threonine, tyrosine.Phenylalanine, tryptophan, and tyrosine are sometimes classified asaromatic amino acids.

An “antigen” refers to a molecule containing one or more epitopes(either linear, conformational or both) that will stimulate a host'simmune-system to make a humoral and/or cellular antigen-specificresponse. The term is used interchangeably with the term “immunogen.”Normally, a B-cell epitope will include at least about 5 amino acids butcan be as small as 3-4 amino acids. A T-cell epitope, such as a CTLepitope, will include at least about 7-9 amino acids, and a helperT-cell epitope at least about 12-20 amino acids. Normally, an epitopewill include between about 7 and 15 amino acids, such as, 9, 10, 12 or15 amino acids. The term includes polypeptides which includemodifications, such as deletions, additions and substitutions (generallyconservative in nature) as compared to a native sequence, so long as theprotein maintains the ability to elicit an immunological response, asdefined herein. These modifications may be deliberate, as throughsite-directed mutagenesis, or may be accidental, such as throughmutations of hosts which produce the antigens.

An “immunological response” to an antigen or composition is thedevelopment in a subject of a humoral and/or a cellular immune responseto an antigen present in the composition of interest. For purposes ofthe present disclosure, a “humoral immune response” refers to an immuneresponse mediated by antibody molecules, while a “cellular immuneresponse” is one mediated by T-lymphocytes and/or other white bloodcells. One important aspect of cellular immunity involves anantigen-specific response by cytolytic T-cells (“CTL”s). CTLs havespecificity for peptide antigens that are presented in association withproteins encoded by the major histocompatibility complex (MHC) andexpressed on the surfaces of cells. CTLs help induce and promote thedestruction of intracellular microbes, or the lysis of cells infectedwith such microbes. Another aspect of cellular immunity involves anantigen-specific response by helper T-cells. Helper T-cells act to helpstimulate the function, and focus the activity of, nonspecific effectorcells against cells displaying peptide antigens in association with MHCmolecules on their surface. A “cellular immune response” also refers tothe production of cytokines, chemokines and other such moleculesproduced by activated T-cells and/or other white blood cells, includingthose derived from CD4+ and CD8+ T-cells. Hence, an immunologicalresponse may include one or more of the following effects: theproduction of antibodies by B-cells; and/or the activation of suppressorT-cells and/or γΔ T-cells directed specifically to an antigen orantigens present in the composition or vaccine of interest. Theseresponses may serve to neutralize infectivity, and/or mediateantibody-complement, or antibody dependent cell cytotoxicity (ADCC) toprovide protection to an immunized host. Such responses can bedetermined using standard immunoassays and neutralization assays, wellknown in the art.

An “immunogenic composition” is a composition that comprises anantigenic molecule where administration of the composition to a subjectresults in the development in the subject of a humoral and/or a cellularimmune response to the antigenic molecule of interest.

“Substantially purified” general refers to isolation of a substance(compound, polynucleotide, protein, polypeptide, polypeptidecomposition) such that the substance comprises the majority percent ofthe sample in which it resides. Typically in a sample a substantiallypurified component comprises 50%, preferably 80%-85%, more preferably90-95% of the sample. Techniques for purifying polynucleotides andpolypeptides of interest are well-known in the art and include, forexample, ion-exchange chromatography, affinity chromatography andsedimentation according to density.

A “coding sequence” or a sequence which “encodes” a selectedpolypeptide, is a nucleic acid molecule which is transcribed (in thecase of DNA) and translated (in the case of mRNA) into a polypeptide invivo when placed under the control of appropriate regulatory sequences(or “control elements”). The boundaries of the coding sequence aredetermined by a start codon at the 5′ (amino) terminus and a translationstop codon at the 3′ (carboxy) terminus. A coding sequence can include,but is not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA,genomic DNA sequences from viral or prokaryotic DNA, and even syntheticDNA sequences. A transcription termination sequence may be located 3′ tothe coding sequence.

Typical “control elements”, include, but are not limited to,transcription promoters, transcription enhancer elements, transcriptiontermination signals, polyadenylation sequences (located 3′ to thetranslation stop codon), sequences for optimization of initiation oftranslation (located 5′ to the coding sequence), and translationtermination sequences, and/or sequence elements controlling an openchromatin structure see e.g., McCaughan et al. (1995) PNAS USA92:5431-5435; Kochetov et al (1998) FEBS Letts. 440:351-355.

A “nucleic acid” molecule can include, but is not limited to,prokaryotic sequences, eukaryotic mRNA, cDNA from eukaryotic mRNA,genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and evensynthetic DNA sequences. The term also captures sequences that includeany of the known base analogs of DNA and RNA.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, a given promoter operably linked to a coding sequence iscapable of effecting the expression of the coding sequence when active.The promoter need not be contiguous with the coding sequence, so long asit functions to direct the expression thereof. Thus, for example,intervening untranslated yet transcribed sequences can be presentbetween the promoter sequence and the coding sequence and the promotersequence can still be considered “operably linked” to the codingsequence.

“Recombinant” as used herein to describe a nucleic acid molecule means apolynucleotide of genomic, cDNA, semisynthetic, or synthetic originwhich, by virtue of its origin or manipulation: (1) is not associatedwith all or a portion of the polynucleotide with which it is associatedin nature; and/or (2) is linked to a polynucleotide other than that towhich it is linked in nature. The term “recombinant” as used withrespect to a protein or polypeptide means a polypeptide produced byexpression of a recombinant polynucleotide. “Recombinant host cells,”“host cells,” “cells,” “cell lines,” “cell cultures,” and other suchterms denoting prokaryotic microorganisms or eukaryotic cell linescultured as unicellular entities, are used interchangeably, and refer tocells which can be, or have been, used as recipients for recombinantvectors or other transfer DNA, and include the progeny of the originalcell which has been transfected. It is understood that the progeny of asingle parental cell may not necessarily be completely identical inmorphology or in genomic or total DNA complement to the original parent,due to accidental or deliberate mutation. Progeny of the parental cellwhich are sufficiently similar to the parent to be characterized by therelevant property, such as the presence of a nucleotide sequenceencoding a desired peptide, are included in the progeny intended by thisdefinition, and are covered by the above terms.

Techniques for determining amino acid sequence “similarity” are wellknown in the art. In general, “similarity” means the exact amino acid toamino acid comparison of two or more polypeptides at the appropriateplace, where amino acids are identical or possess similar chemicaland/or physical properties such as charge or hydrophobicity. A so-termed“percent similarity” then can be determined between the comparedpolypeptide sequences. Techniques for determining nucleic acid and aminoacid sequence identity also are well known in the art and includedetermining the nucleotide sequence of the mRNA for that gene (usuallyvia a cDNA intermediate) and determining the amino acid sequence encodedthereby, and comparing this to a second amino acid sequence. In general,“identity” refers to an exact nucleotide to nucleotide or amino acid toamino acid correspondence of two polynucleotides or polypeptidesequences, respectively.

Two or more polynucleotide sequences can be compared by determiningtheir “percent identity.” Two or more amino acid sequences likewise canbe compared by determining their “percent identity.” The percentidentity of two sequences, whether nucleic acid or peptide sequences, isgenerally described as the number of exact matches between two alignedsequences divided by the length of the shorter sequence and multipliedby 100. An approximate alignment for nucleic acid sequences is providedby the local homology algorithm of Smith and Waterman, Advances inApplied Mathematics 2:482-489 (1981). This algorithm can be extended touse with peptide sequences using the scoring matrix developed byDayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5suppl. 3:353-358, National Biomedical Research Foundation, Washington,D.C., USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763(1986). Suitable programs for calculating the percent identity orsimilarity between sequences are generally known in the art.

A “vector” is capable of transferring gene sequences to target cells(e.g., bacterial plasmid vectors, viral vectors, non-viral vectors,particulate carriers, and liposomes). Typically, “vector construct,”“expression vector,” and “gene transfer vector,” mean any nucleic acidconstruct capable of directing the expression of one or more sequencesof interest in a host cell. Thus, the term includes cloning andexpression vehicles, as well as viral vectors. The term is usedinterchangeable with the terms “nucleic acid expression vector” and“expression cassette.”

By “subject” is meant any member of the subphylum chordata, including,without limitation, humans and other primates, including non-humanprimates such as chimpanzees and other apes and monkey species; farmanimals such as cattle, sheep, pigs, goats and horses; domestic mammalssuch as dogs and cats; laboratory animals including rodents such asmice, rats and guinea pigs; birds, including domestic, wild and gamebirds such as chickens, turkeys and other gallinaceous birds, ducks,geese, and the like. The term does not denote a particular age. Thus,both adult and newborn individuals are intended to be covered. Thesystem described above is intended for use in any of the abovevertebrate species, since the immune systems of all of these vertebratesoperate similarly.

By “pharmaceutically acceptable” or “pharmacologically acceptable” ismeant a material which is not biologically or otherwise undesirable,i.e., the material may be administered to an individual in a formulationor composition without causing any unacceptable biological effects orinteracting in a deleterious manner with any of the components of thecomposition in which it is contained.

As used herein, “treatment” refers to any of (i) the prevention ofinfection or reinfection, as in a traditional vaccine, (ii) thereduction or elimination of symptoms, and (iii) the substantial orcomplete elimination of the pathogen in question. Treatment may beeffected prophylactically (prior to infection) or therapeutically(following infection).

As used herein the term “adjuvant” refers to a compound that, when usedin combination with a specific immunogen (e.g. a VLP) in a formulation,will augment or otherwise alter or modify the resultant immune response.Modification of the immune response includes intensification orbroadening the specificity of either or both antibody and cellularimmune responses. Modification of the immune response can also meandecreasing or suppressing certain antigen-specific immune responses.

As used herein an “effective dose” generally refers to that amount ofVLPs of the invention sufficient to induce immunity, to prevent and/orameliorate an infection or to reduce at least one symptom of aninfection and/or to enhance the efficacy of another dose of a VLP. Aneffective dose may refer to the amount of VLPs sufficient to delay orminimize the onset of an infection. An effective dose may also refer tothe amount of VLPs that provides a therapeutic benefit in the treatmentor management of an infection. Further, an effective dose is the amountwith respect to VLPs of the invention alone, or in combination withother therapies, that provides a therapeutic benefit in the treatment ormanagement of an infection. An effective dose may also be the amountsufficient to enhance a subject's (e.g., a human's) own immune responseagainst a subsequent exposure to an infectious agent. Levels of immunitycan be monitored, e.g., by measuring amounts of neutralizing secretoryand/or serum antibodies, e.g., by plaque neutralization, complementfixation, enzyme-linked immunosorbent, or microneutralization assay. Inthe case of a vaccine, an “effective dose” is one that prevents diseaseand/or reduces the severity of symptoms.

As used herein, the term “effective amount” refers to an amount of VLPsnecessary or sufficient to realize a desired biologic effect. Aneffective amount of the composition would be the amount that achieves aselected result, and such an amount could be determined as a matter ofroutine experimentation by a person skilled in the art. For example, aneffective amount for preventing, treating and/or ameliorating aninfection could be that amount necessary to cause activation of theimmune system, resulting in the development of an antigen specificimmune response upon exposure to VLPs of the invention. The term is alsosynonymous with “sufficient amount.”

As used herein, the term “multivalent” refers to VLPs which havemultiple antigenic proteins against multiple types or strains ofinfectious agents.

As used herein the term “immune stimulator” refers to a compound thatenhances an immune response via the body's own chemical messengers(cytokines). These molecules comprise various cytokines, lymphokines andchemokines with immunostimulatory, immunopotentiating, andpro-inflammatory activities, such as interferons, interleukins (e.g.,IL-1, IL-2, IL-3, L-4, IL-12, IL-13); growth factors (e.g.,granulocyte-macrophage (GM)-colony stimulating factor (CSF)); and otherimmunostimulatory molecules, such as macrophage inflammatory factor,Flt3 ligand, B7.1; B7.2, etc. The immune stimulator molecules can beadministered in the same formulation as VLPs of the invention, or can beadministered separately. Either the protein or an expression vectorencoding the protein can be administered to produce an immunostimulatoryeffect.

As used herein the term “protective immune response” or “protectiveresponse” refers to an immune response mediated by antibodies against aninfectious agent, which is exhibited by a vertebrate (e.g., a human),that prevents or ameliorates an infection or reduces at least onesymptom thereof. VLPs of the invention can stimulate the production ofantibodies that, for example, neutralize infectious agents, blocksinfectious agents from entering cells, blocks replication of saidinfectious agents, and/or protect host cells from infection anddestruction. The term can also refer to an immune response that ismediated by T-lymphocytes and/or other white blood cells against aninfectious agent, exhibited by a vertebrate (e.g., a human), thatprevents or ameliorates flavivirus infection or reduces at least onesymptom thereof.

As use herein, the term “antigenic formulation” or “antigeniccomposition” refers to a preparation which, when administered to avertebrate, e.g. a mammal, will induce an immune response.

As used herein, the term “vaccine” refers to a formulation whichcontains VLPs of the present invention, which is in a form that iscapable of being administered to a vertebrate and which induces aprotective immune response sufficient to induce immunity to preventand/or ameliorate an infection and/or to reduce at least one symptom ofan infection and/or to enhance the efficacy of another dose of VLPs.Typically, the vaccine comprises a conventional saline or bufferedaqueous solution medium in which the composition of the presentinvention is suspended or dissolved. In this form, the composition ofthe present invention can be used conveniently to prevent, ameliorate,or otherwise treat an infection. Upon introduction into a host, thevaccine is able to provoke an immune response including, but not limitedto, the production of antibodies and/or cytokines and/or the activationof cytotoxic T cells, antigen presenting cells, helper T cells,dendritic cells and/or other cellular responses.

General Overview

This invention describes the formation of biological particles (e.g.,VLPs) that mimic the structure in size, morphology and biochemicalcomposition of native Zika viruses and other flaviviruses; however, theyare devoid of a fully competent viral genome and therefore unable tocause infection or disease. The lack of viral genome and lack ofinfectivity of the flavivirus (Zika) VLPs eliminate the need of chemicalinactivation better preserving therefore their structures, proteinconformations and antigenic properties enhancing immunogenicity andpotency as vaccine. These biological mimics are identified as virus-likeparticles (VLPs). VLPs are assembled using genetic informationcomprising segments of the virus genome encoding selected proteins thatmay include but not limited to structural and non-structural protein. Asshown in FIG. 1, the viral sequences in DNA form can be organized in asingle transcription unit (segment) that expresses a single polypeptideor in separate transcription units (segments) each one expressing asingle protein.

Virus-Like Particles

The present disclosure relates to flavivirus VLPs, which VLPs carry ontheir surfaces one or more modified antigenic flavivirus proteins. ThisVLP, alone or in combination with one or more additional VLPs and/oradjuvants, stimulates an immune response that protects againstflavivirus infection.

In one embodiment of the invention, the structural proteins of interestcomprise CprME, which after expression leads to the formation of VLPs.In order to enhance assembly and release of these VLPs from theproducing cells, non-structural proteins may be co-expressed with thestructural proteins (e.g. NS2B/NS3). Exemplary wild-type and mutantCprME nucleotide and amino acid sequences are shown in the “Sequences”Section below (e.g. Zika CprME wild type nucleotide sequence, SEQ ID NO:20 and codon optimized SEQ ID NO:21 and the amino acid sequence is shownin SEQ ID NO:22). Exemplary of Zika NS2B/NS3 full-length nucleotidesequence (e.g. SEQ ID NO:23) and amino acid sequence (e.g. SEQ ID NO:24). Also, examples of truncated and codon optimized nucleotide sequenceof Zika NS2B/NS3 is shown in SEQ ID NO: 25 and its corresponding aminoacid sequence is described in SEQ ID NO: 26. It will be apparentproteins from any Zika serotype or strain can be used in thecompositions described herein, for example by alignment with theexemplary Zika sequences disclosed herein.

In any of the VLPs and methods described herein, the non-structuralproteins may comprise either a full-length NS3 segment or a truncatedversion of the segment containing the protease domain, which islocalized at the amino terminal of the polypeptide (e.g. truncatedversion of NS3 comprising aa 1 to aa 181). Exemplary sequences are shownbelow (e.g. full length amino acid sequence of dengue NS3, SEQ ID NO:27,and truncated aa 1 to aa 181 and mutated, SEQ ID NO:13).

In any of the VLPs or methods described herein, the non-structuralprotein segment comprising the full length or truncated form of NS3 isco-expressed with the full length or truncated forms of NS2B. Exemplarywild-type and mutant NS3 and/or NS2B nucleotide and amino acid sequencesare shown in the “Sequences” section below. (e.g. an example of wildtype nucleotide sequence of dengue NS2B/NS3 is shown in SEQ ID NO: 6 andamino acid sequence described in SEQ ID NO: 7). Modified dengue NS2B/NS3(e.g., truncated, mutated and codon optimized) nucleotide sequence isshown in SEQ ID NO: 8 and amino acid sequence shown in SEQ ID NO: 9.When NS2B or NS3 are used as single proteins the nucleotide sequence isderived from SEQ ID NO: 8 resulting in NS2B nucleotide sequence (e.g.SEQ ID NO: 10) and amino acid sequence (SEQ ID NO: 11) and NS3nucleotide sequence (e.g. SEQ ID NO: 12) and amino acid (e.g. SEQ ID NO:13). In certain embodiments, the structural and non-structural proteinsmay be genetically linked, (as shown in FIG. 2). This is a non-limitingexample of a single segment with a defined order using a linker that mayencode. Additional exemplary sequences that may be used include but arenot limited to a sequence of different portions of the NS1, NS2A and/orNS2B proteins as follows:

Linker 1: amino acids (aa) 1 to 8 or 9 or 10 of NS1 (e.g. nucleotidesequence SEQ ID NO: 14 and amino acid sequence SEQ ID NO: 15) connectedto a portion of NS2A comprising of aa 186 or 187 or 188 or 189 to aa 218of NS2A (e.g. nucleotide sequence SEQ ID NO: 16 and amino acid sequenceSEQ ID NO: 17).

Linker 2: aa 1 to 8 or 9 or 10 of NS1 (SEQ ID NO: 14) connected to afirst portion of NS2A comprising aa 1 to 24 or 25 or 26 or 27 or 28 or29 or 30 or 31 or 32 connected to a second portion of NS2A comprising ofaa 186 or 187 or 188 or 189 to 218 of NS2A (SEQ ID NO: 16).

Linker 3: aa 1 to 8 or 9 or 10 of NS1 (SEQ ID NO: 14) connected to thesecond transmembrane domain of NS2B (e.g. nucleotide sequence SEQ ID NO:10 and amino acid sequence SEQ ID NO: 11)

Linker 4: aa 1 to 8 or 9 or 10 of NS1 (SEQ ID NO: 14) connected to thefirst transmembrane domain of NS2A (Amino acid 51 to 100 of nucleotidesequence, SEQ ID NO: 16 and amino acid sequence SEQ ID NO: 17)

Linker 5: aa 1 to 8 or 9 or 10 of NS1 (SEQ ID NO: 14) connected to the Cterminal portion of NS2B comprising the second transmembrane domain tothe end of the protein (e.g. nucleotide sequence SEQ ID NO: 10 and aminoacid sequence SEQ ID NO:11).

In still further embodiments, the positional order of the structural andnon-structural segments may be inverted (with respect to each other)where the non-structural segment is genetically linked to the structuralsegment with or without a connecting linker. For example, thenon-structural protein may include a full length NS2B and a full NS3genetically linked directly to the structural proteins CprME.Alternatively, the non-structural proteins may comprise a truncatedvariant of NS3 in which the helicase domain is deleted but the proteasedomain and its recognizable self-cleavage site located at the carboxylterminal of the protein are preserved. The self-cleavage site serves asone example of a linker that may be used to connect the non-structuraland structural proteins.

In still further embodiments, the VLPs and methods described herein mayinclude changes in the sequence of the structural and non-structuralproteins (e.g., modifications to the nucleotide sequence which result inamino acid modifications), which can be used to enhance the formationand release of the VLP from the producing cells.

For example, the furin protease cleavage site between pr and M proteinmay be modified to enhance furin activity. The recognition consensussequence is defined as R-Xaa-L/R-R where amino acids immediately priorto the cleavage site specifies positions P1 (R), P2 L/R, P3 (Xaa) and P4(R). The naturally occurring acid residue in the cleavage site atposition P3 can be substituted, but not limited to, by a hydrophobicresidue. Exemplary sequences are shown below in “Sequences” section (e.gSEQ ID NO: 4)

In other embodiments, one or more helices of the E protein may bemodified to enhance VLP assemble and release (Purdy D E, Chang G-JJ,2005, Secretion of noninfectious dengue virus-like particles andidentification of amino acids in the stem region involved inintracellular retention of envelope protein, Virology 333(2): 239-250).

For example, the amphipathic helix 1 in the stem domain of the E proteinmay be modified to enhance the hydrophobic properties of one side ofhelix.

In one exemplary embodiments of this modification: one, two, three ormore amino acids in the hydrophobic side of the helix are substituted.These substitutions may occur at positions 398, 401 and/or 412,including but not limited to I398L, I398M, I398V, I398A or M401A, M401L,M401V, M401I, or M412A, M412L, M412V, M412I.

In other embodiments, the helix 1 and/or helix 2 are exchanged with thehelix sequences derived from other flaviviruses or other viruses andsources.

In other embodiments, the NS3 protease active site may be modified inorder to enhance its enzymatic activity. Non-limiting examples of suchmodifications include the substitution of amino acid leucine at position115 (e.g., with an amino acid with a shorter side chain such as glycineor alanine).

In still further embodiments, to boost the protease cleavage between prand M by furin and therefore increase the amount of mature particles,additional furin enzymes may be furnished throughout the VLP productionusing any of the methods described below:

One version of this example may include the addition of recombinantfurin protein to the VLPs producing cells culture medium.

A second version of this example: an expression plasmid carrying thefurin gene as a single transcription unit (segment) may beco-transfected with one or more plasmids expressing the structural andnon-structural genes. Another method may include an expression plasmidcarrying a single transcription unit (segment) where the furin gene isgenetically linked to either structural or non-structural protein orboth.

A third version of this example: a cell line stably transfected andselected for its constitutive expression of furin, may be used in VLPproduction. Alternatively, the furin gene is stably transfected and/orinducible in a cell line already modified to constitutively produceVLPs.

VLP Production

The production of VLPs as described herein may be achieved by anysuitable method, including but not limited to transient and/or stableexpression of the structural and/or non-structural genes in a suspensionculture of eukaryotic cells, typically requiring a period of continuedcell culture after which the VLPs are harvested from the culture medium.The VLPs produced as described herein are conveniently prepared usingstandard recombinant techniques. Polynucleotides encoding theVLP-forming protein(s) are introduced into a host cell and, when theproteins are expressed in the cell, they assembly into VLPs.

Polynucleotide sequences coding for molecules (proteins) that formand/or are incorporated into the VLPs can be obtained using recombinantmethods, such as by screening cDNA and genomic libraries from cellsexpressing the gene, or by deriving the gene from a vector known toinclude the same. For example, plasmids which contain sequences thatencode naturally occurring or altered cellular products may be obtainedfrom a depository such as the A.T.C.C., or from commercial sources.Plasmids containing the nucleotide sequences of interest can be digestedwith appropriate restriction enzymes, and DNA fragments containing thenucleotide sequences can be inserted into a gene transfer vector usingstandard molecular biology techniques.

Alternatively, cDNA sequences may be obtained from cells which expressor contain the sequences, using standard techniques, such as phenolextraction and PCR of cDNA or genomic DNA. See, e.g., Sambrook et al.,supra, for a description of techniques used to obtain and isolate DNA.Briefly, mRNA from a cell which expresses the gene of interest can bereverse transcribed with reverse transcriptase using oligo-dT or randomprimers. The single stranded cDNA may then be amplified by PCR (see U.S.Pat. Nos. 4,683,202, 4,683,195 and 4,800,159, see also PCR Technology:Principles and Applications for DNA Amplification, Erlich (ed.),Stockton Press, 1989)) using oligonucleotide primers complementary-tosequences on either side of desired sequences.

The nucleotide sequence of interest can also be produced synthetically,rather than cloned, using a DNA synthesizer (e.g., an Applied BiosystemsModel 392 DNA Synthesizer, available from ABI, Foster City, Calif.). Thenucleotide sequence can be designed with the appropriate codons for theexpression product desired. The complete sequence is assembled fromoverlapping oligonucleotides prepared by standard methods and assembledinto a complete coding sequence. See, e.g., Edge (1981) Nature 292:756;Nambair et al. (1984) Science 223:1299; Jay et al. (1984) J. Biol. Chem.259:6311.

Preferably, the sequences employed to form flavivirus VLPs exhibitbetween about 60% to 80% (or any value therebetween including 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78% and 79%) sequence identity to a naturally occurring flaviviruspolynucleotide sequence and more preferably the sequences exhibitbetween about 80% and 100% (or any value therebetween including 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% and 99%) sequence identity to a naturally occurringpolynucleotide sequence.

Any of the sequences described herein may further include additionalsequences. For example, to further to enhance vaccine potency, hybridmolecules are expressed and incorporated into the sub-viral structure.These hybrid molecules are generated by linking, at the DNA level, thesequences coding for the protein genes with sequences coding for anadjuvant or immuno-regulatory moiety. During sub-viral structureformation, these hybrid proteins are incorporated into or onto theparticle. The incorporation of one or more polypeptide immunomodulatorypolypeptides (e.g., adjuvants describe in detail below) into thesequences described herein into the VLP may enhance potency andtherefore reduces the amount of antigen required for stimulating aprotective immune response. Alternatively, as described below, one ormore additional molecules (polypeptide or small molecules) may beincluded in the VLP-containing compositions after production of the VLPfrom the sequences described herein.

These sub-viral structures do not contain infectious viral nucleic acidsand they are not infectious eliminating the need for chemicalinactivation. Absence of chemical treatment preserves native epitopesand protein conformations enhancing the immunogenic characteristics ofthe vaccine.

The sequences described herein can be operably linked to each other inany combination. For example, one or more sequences may be expressedfrom the same promoter and/or from different promoters. As describedbelow, sequences may be included on one or more vectors. Non-limitingexamples of vectors that can be used to express sequences that assembleinto VLPs as described herein include viral-based vectors (e.g.,retrovirus, adenovirus, adeno-associated virus, lentivirus), baculovirusvectors (see, Examples), plasmid vectors, non-viral vectors, mammaliansvectors, mammalian artificial chromosomes (e.g., liposomes, particulatecarriers, etc.) and combinations thereof. The expression vector(s)typically contain(s) coding sequences and expression control elementswhich allow expression of the coding regions in a suitable host.Enhancer elements may also be used herein to increase expression levelsof the mammalian constructs.

Vaccine formulation is accomplished according to standard procedures,for example as shown in FIG. 3.

Monovalent

Different exemplary strategies are described to assemble monovalent VLPsincluding:

1. Use homologous clades/antigenic variants for both structural andnon-structural proteins

2. Use heterologous clades between the structural proteins andnon-structural proteins with the exception of the viral cleavage sitewithin the sequence of the C protein that matches the clade/antigenicvariant of the non-structural proteins

3. Use heterologous clades between the structural proteins andnon-structural proteins with the exception of the cytoplasmic domainincluding the viral protease cleavage site of the C protein, whichmatches the serotype of the non-structural proteins

Bivalent

Two exemplary strategies to generate a bivalent vaccine:

1. Blending two monovalent VLP in a single formulation.

-   -   1a. Use homologous clades/antigenic variants for both structural        and non-structural proteins    -   1b. Use heterologous clades/antigenic variants between the        structural proteins and non-structural proteins with the        exception of the viral cleavage site within the sequence of the        C protein that matches the serotype of the non-structural        proteins    -   1c. Use heterologous clades/antigenic variants between the        structural proteins and non-structural proteins with the        exception of the cytoplasmic domain including the viral protease        cleavage site of the C protein, which matches the serotype of        the non-structural proteins (e.g. SEQ. ID NO: 35 and SEQ ID NO:        36, SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 42, SEQ ID NO:        43)

2. Assembly of a single bivalent particle VLP. Alternative exemplaryapproach may be used to build these structures:

-   -   2a. Co-expression of two heterologous set of both structural and        non-structural proteins    -   2b. Co-expression of two heterologous structural proteins        containing the same viral cleavage site within the sequence of        the C protein together with non-structural proteins that        recognized this viral cleavage site.    -   2c. Co-expression of two heterologous structural proteins that        share the analogous cytoplasmic domain sequence including the        viral protease cleavage site of the C protein together with        non-structural proteins that recognize this viral cleavage site

Multivalent

Several exemplary approaches may be used to create multivalent vaccineformulations:

1. Blending of several single monovalent VLPs as described in themonovalent sections 1, 2 and 3

2. Blending of two single particle bivalents in a sole combination.Assembly of the single particle bivalent is as described in section 2a,2b and 2c.

3. Assembly of single multivalent particle: To assemble this particle

-   -   3a. Co-expression of several heterologous sets of both        structural and non-structural proteins    -   3b. Co-expression of several heterologous structural proteins        containing the same viral cleavage site within the sequence of        the C protein together with non-structural proteins that        recognize this viral cleavage site    -   3c. Co-expression of several heterologous structural proteins        that share the analogous cytoplasmic domain sequence including        the viral protease cleavage site of the C protein together with        non-structural proteins that recognize this viral cleavage site.

Furthermore, combination vaccine can be created by blending in a singleformulation monovalent, bivalent or multivalent compositions of adisease-causing virus (e.g. dengue) with composition of anotherdisease-causing virus such as Zika or chikungunya.

The utility of the VLPs include, but it is not limited to, thegeneration of immunogenic (vaccine) compositions that when administeredto humans are able to treat and/or prevent flavivirus (dengue, Zika)infection, including treatment and/or prevention of infection with oneand/or more flavivirus virus clades/antigenic variants or serotypes.Flavivirus VLPs may be used in combination with other flavivirus VLPs(e.g., Zika with dengue, and/or Alpha chikungunya, etc.), including VLPsthat include one or more antigenic determinants from two or moreflaviviruses and combinations of VLPs that include one or more antigenicdeterminants from one, two or more flaviviruses with VLPs that includesone or more antigenic determinants from one, two or more flaviviruses(e.g., monovalent, bivalent or multivalent Zika VLP in a pharmaceuticalcomposition with monovalent, bivalent or multivalent Zika, dengue and/orchikungunya VLP). Other utility of the Zika VLP relates to diagnosticand therapeutic applications.

Suitable host cells for producing VLPs as described herein include, butare not limited to, bacterial, mammalian, baculovirus/insect, yeast,plant and Xenopus cells. For example, a number of mammalian cell linesare known in the art and include primary cells as well as immortalizedcell lines available from the American Type Culture Collection(A.T.C.C.), such as, but not limited to, MDCK, BHK, VERO, MRC-5, WI-38,HT1080, 293, 293T, RD, COS-7, CHO, Jurkat, HUT, SUPT, C8166,MOLT4/clone8, MT-2, MT-4, H9, PM1, CEM, myeloma cells (e.g., SB20 cells)and CEMX174 (such cell lines are available, for example, from theA.T.C.C.).

The immunogenicity of VLP vaccines may be affected by the structuralconformation of the E protein displayed on the particles' surface.Changing the temperature of the fermentation process may alter thisconformation. In one embodiment, the VLPs are produced at lowertemperature (31° C., plus or minus 3 degrees centigrade) than thestandard temperature of fermentation of 37° C. VLPs produced at thelower temperature when administered as vaccine may induce higherneutralizing antibody titers than those produced at 37° C.

The VLPs as described herein may be purified following production.Non-limiting examples of suitable purification (isolation) from the cellculture medium procedures include using centrifugation and/or gradientcentrifugation under suitable conditions. Other methods of purificationmay include sequential steps of filtration and/or chromatographyprocedures including ion exchange, affinity, size exclusion and/orhydrophobic interaction chemistries.

Cell lines expressing one or more of the sequences described above canreadily be generated given the disclosure provided herein by stablyintegrating one or more expression vector constructs encoding theproteins of the VLP. The promoter regulating expression of the stablyintegrated flavivirus sequences (s) may be constitutive or inducible.Thus, a cell line can be generated in which one or more structuralproteins are stably integrated such that, upon introduction of thesequences described herein (e.g., hybrid proteins) into a host cell andexpression of the proteins encoded by the polynucleotides,non-replicating viral particles that present antigenic glycoproteins areformed.

In certain embodiments, a mammalian cell line that stably expressed twoor more antigenically distinct flavivirus proteins is generated.Sequences encoding structural and/or non-structural proteins can beintroduced into such a cell line to produce VLPs as described herein.Alternatively, a cell line that stably produces structural proteins canbe generated and sequences encoding the antigenic flavivirus protein(s)from the selected strain(s)/serotype(s)/clade(s) introduced into thecell line, resulting in production of VLPs presenting the desiredantigenic glycoproteins.

The parent cell line from which an VLP-producer cell line is derived canbe selected from any cell described above, including for example,mammalian, insect, yeast, bacterial cell lines. In a preferredembodiment, the cell line is a mammalian cell line (e.g., 293, RD,COS-7, CHO, BHK, MDCK, MDBK, MRC-5, VERO, HT1080, and myeloma cells).Production of VLPs using mammalian cells provides (i) VLP formation;(ii) correct post translation modifications (glycosylation,palmitylation) and budding; (iii) absence of non-mammalian cellcontaminants and (iv) ease of purification.

In addition to creating cell lines, flavivirus-encoding sequences mayalso be transiently expressed in host cells. Suitable recombinantexpression host cell systems include, but are not limited to, bacterial,mammalian, baculovirus/insect, vaccinia, Semliki Forest virus (SFV),Alphaviruses (such as, Sindbis, Venezuelan Equine Encephalitis (VEE)),Retrovirus vectors (lentivirus), mammalian, yeast and Xenopus expressionsystems, well known in the art. Particularly preferred expressionsystems are mammalian cell lines, vaccinia, Sindbis, insect and yeastsystems.

Many suitable expression systems are commercially available, including,for example, the following: baculovirus expression (Reilly, P. R., etal., BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL (1992); Beames,et al., Biotechniques 11:378 (1991); Pharmingen; Clontech, Palo Alto,Calif.)), vaccinia expression systems (Earl, P. L., et al., “Expressionof proteins in mammalian cells using vaccinia” In Current Protocols inMolecular Biology (F. M. Ausubel, et al. Eds.), Greene PublishingAssociates & Wiley Interscience, New York (1991); Moss, B., et al., U.S.Pat. No. 5,135,855, issued Aug. 4, 1992), expression in bacteria(Ausubel, F. M., et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, JohnWiley and Sons, Inc., Media Pa.; Clontech), expression in yeast(Rosenberg, S. and Tekamp-Olson, P., U.S. Pat. No. RE35,749, issued,Mar. 17, 1998, herein incorporated by reference; Shuster, J. R., U.S.Pat. No. 5,629,203, issued May 13, 1997, herein incorporated byreference; Gellissen, G., et al., Antonie Van Leeuwenhoek, 62(1-2):79-93(1992); Romanos, M. A., et al., Yeast 8(6):423-488 (1992); Goeddel, D.V., Methods in Enzymology 185 (1990); Guthrie, C., and G. R. Fink,Methods in Enzymology 194 (1991)), expression in mammalian cells(Clontech; Gibco-BRL, Ground Island, N.Y.; e.g., Chinese hamster ovary(CHO) cell lines (Haynes, J., et al., Nuc. Acid. Res. 11:687-706 (1983);1983, Lau, Y. F., et al., Mol. Cell. Biol. 4:1469-1475 (1984); Kaufman,R. J., “Selection and coamplification of heterologous genes in mammaliancells,” in Methods in Enzymology, vol. 185, pp 537-566. Academic Press,Inc., San Diego Calif. (1991)), and expression in plant cells (plantcloning vectors, Clontech Laboratories, Inc., Palo-Alto, Calif., andPharmacia LKB Biotechnology, Inc., Pistcataway, N.J.; Hood, E., et al.,J. Bacteriol. 168:1291-1301 (1986); Nagel, R., et al., FEMS Microbiol.Lett. 67:325 (1990); An, et al., “Binary Vectors”, and others in PlantMolecular Biology Manual A3:1-19 (1988); Miki, B. L. A., et al., pp.249-265, and others in Plant DNA Infectious Agents (Hohn, T., et al.,eds.) Springer-Verlag, Wien, Austria, (1987); Plant Molecular Biology:Essential Techniques, P. G. Jones and J. M. Sutton, New York, J. Wiley,1997; Miglani, Gurbachan Dictionary of Plant Genetics and MolecularBiology, New York, Food Products Press, 1998; Henry, R. J., PracticalApplications of Plant Molecular Biology, New York, Chapman & Hall,1997).

When expression vectors containing the altered genes that code for theproteins required for sub-viral structure vaccine formation areintroduced into host cell(s) and subsequently expressed at the necessarylevel, the sub-viral structure vaccine assembles and is then releasedfrom the cell surface into the culture media (FIG. 7).

Depending on the expression system and host selected, the VLPs areproduced by growing host cells transformed by an expression vector underconditions whereby the particle-forming polypeptide(s) is (are)expressed and VLPs can be formed. The selection of the appropriategrowth conditions is within the skill of the art. If the VLPs are formedand retained intracellularly, the cells are then disrupted, usingchemical, physical or mechanical means, which lyse the cells yet keepthe VLPs substantially intact. Such methods are known to those of skillin the art and are described in, e.g., Protein PurificationApplications: A Practical Approach, (E. L. V. Harris and S. Angal, Eds.,1990). Alternatively, VLPs may be secreted and harvested from thesurrounding culture media.

The particles are then isolated (or substantially purified) usingmethods that preserve the integrity thereof, such as, by densitygradient centrifugation, e.g., sucrose, potassium tartrate or Iodixanolgradients, PEG-precipitation, pelleting, and the like (see, e.g.,Kirnbauer et al. J. Virol. (1993) 67:6929-6936), as well as standardpurification techniques including, e.g., ion exchange and gel filtrationchromatography, tangential filtration, etc.

Compositions

VLPs produced as described herein can be used to elicit an immuneresponse when administered to a subject. As discussed above, the VLPscan comprise a variety of antigens (e.g., one or more modifiedflavivirus antigens from one or more flaviviruses and/or one or morestrains, serotypes, clades or isolates of a particular flavivirus).Purified VLPs can be administered to a vertebrate subject, usually inthe form of vaccine compositions. Combination vaccines may also be used,where such vaccines contain, for example, other proteins derived fromother flaviviruses or other organisms and/or gene delivery vaccinesencoding such antigens.

VLP immune-stimulating (or vaccine) compositions can include variousexcipients, adjuvants, carriers, auxiliary substances, modulatingagents, and the like. The immune stimulating compositions will includean amount of the VLP/antigen sufficient to mount an immunologicalresponse. An appropriate effective amount can be determined by one ofskill in the art. Such an amount will fall in a relatively broad rangethat can be determined through routine trials and will generally be anamount on the order of about 0.1 μg to about 10 (or more) mg, morepreferably about 1 μg to about 300 μg, of VLP/antigen.

Sub-viral structure vaccines are purified from the cell culture mediaand formulated with the appropriate buffers and additives, such as a)preservatives or antibiotics; b) stabilizers, including proteins ororganic compounds; c) adjuvants or immuno-modulators for enhancingpotency and modulating immune responses (humoral and cellular) to thevaccine; or d) molecules that enhance presentation of vaccine antigensto specifics cell of the immune system. This vaccine can be prepared ina freeze-dried (lyophilized) form in order to provide for appropriatestorage and maximize the shelf-life of the preparation. This will allowfor stock piling of vaccine for prolonged periods of time maintainingimmunogenicity, potency and efficacy.

A carrier is optionally present in the compositions described herein.Typically, a carrier is a molecule that does not itself induce theproduction of antibodies harmful to the individual receiving thecomposition. Suitable carriers are typically large, slowly metabolizedmacromolecules such as proteins, polysaccharides, polylactic acids,polyglycollic acids, polymeric amino acids, amino acid copolymers, lipidaggregates (such as oil droplets or liposomes), and inactive virusparticles. Examples of particulate carriers include those derived frompolymethyl methacrylate polymers, as well as microparticles derived frompoly(lactides) and poly(lactide-co-glycolides), known as PLG. See, e.g.,Jeffery et al., Pharm. Res. (1993) 10:362-368; McGee J P, et al., JMicroencapsul. 14(2):197-210, 1997; O'Hagan D T, et al., Vaccine11(2):149-54, 1993. Such carriers are well known to those of ordinaryskill in the art.

Additionally, these carriers may function as immunostimulating agents(“adjuvants”). Exemplary adjuvants include, but are not limited to: (1)aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate,aluminum sulfate, etc.; (2) oil-in-water emulsion formulations (with orwithout other specific immunostimulating agents such as muramyl peptides(see below) or bacterial cell wall components), such as for example (a)MF59 (International Publication No. WO 90/14837), containing 5%Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing variousamounts of MTP-PE (see below), although not required) formulated intosubmicron particles using a microfluidizer such as Model 110Ymicrofluidizer (Microfluidics, Newton, Mass.), (b) SAF, containing 10%Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP(see below) either microfluidized into a submicron emulsion or vortexedto generate a larger particle size emulsion, and (c) Ribi™ adjuvantsystem (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene,0.2% Tween 80, and one or more bacterial cell wall components from thegroup consisting of monophosphorylipid A (MPL), trehalose dimycolate(TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detoxu); (3)saponin adjuvants, such as Stimulon™. (Cambridge Bioscience, Worcester,Mass.) may be used or particle generated therefrom such as ISCOMs(immunostimulating complexes); (4) Complete Freunds Adjuvant (CFA) andIncomplete Freunds Adjuvant (IFA); (5) cytokines, such as interleukins(IL-1, IL-2, etc.), macrophage colony stimulating factor (M-CSF), tumornecrosis factor (TNF), beta chemokines (MIP, 1-alpha, 1-beta Rantes,etc.); (6) detoxified mutants of a bacterial ADP-ribosylating toxin suchas a cholera toxin (CT), a pertussis toxin (PT), or an E. coliheat-labile toxin (LT), particularly LT-K63 (where lysine is substitutedfor the wild-type amino acid at position 63) LT-R72 (where arginine issubstituted for the wild-type amino acid at position 72), CT-S109 (whereserine is substituted for the wild-type amino acid at position 109), andPT-K9/G129 (where lysine is substituted for the wild-type amino acid atposition 9 and glycine substituted at position 129) (see, e.g.,International Publication Nos. WO93/13202 and WO92/19265); and (7) othersubstances that act as immunostimulating agents to enhance theeffectiveness of the composition.

Muramyl peptides include, but are not limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acteyl-normuramyl-L-alanyl-D-isogluatme (nor-MDP),N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy)-ethylamine(MTP-PE), etc.

Examples of suitable immunomodulatory molecules for use herein includeadjuvants described above and the following: IL-1 and IL-2 (Karupiah etal. (1990) J. Immunology 144:290-298, Weber et al. (1987) J. Exp. Med.166:1716-1733, Gansbacher et al. (1990) J. Exp. Med. 172:1217-1224, andU.S. Pat. No. 4,738,927-); IL-3 and IL-4 (Tepper et al. (1989) Cell57:503-512, Golumbek et al. (1991) Science 254:713-716, and U.S. Pat.No. 5,017,691); IL-5 and IL-6 (Brakenhof et al. (1987) J. Immunol.139:4116-4121, and International Publication No. WO 90/06370); IL-7(U.S. Pat. No. 4,965,195); IL-8, IL-9, IL-10, IL-11, IL-12, and IL-13(Cytokine Bulletin, Summer 1994); IL-14 and IL-15; alpha interferon(Finter et al. (1991) Drugs 42:749-765, U.S. Pat. Nos. 4,892,743 and4,966,843, International Publication No. WO 85/02862, Nagata et al.(1980) Nature 284:316-320, Familletti et al. (1981) Methods in Enz.78:387-394, Twu et al. (1989) Proc. Natl. Acad. Sci. USA 86:2046-2050,and Faktor et al. (1990) Oncogene 5:867-872); β-interferon (Seif et al.(1991) J. Virol. 65:664-671); γ-interferons (Watanabe et al. (1989)Proc. Natl. Acad. Sci. USA 86:9456-9460, Gansbacher et al. (1990) CancerResearch 50:7820-7825, Maio et al. (1989) Can. Immunol. Immunother.30:34-42, and U.S. Pat. Nos. 4,762,791 and 4,727,138); G-CSF (U.S. Pat.Nos. 4,999,291 and 4,810,643); GM-CSF (International Publication No. WO85/04188); tumor necrosis factors (TNFs) (Jayaraman et al. (1990) J.Immunology 144:942-951); CD3 (Krissanen et al. (1987) Immunogenetics26:258-266); ICAM-1 (Altman et al. (1989) Nature 338:512-514, Simmons etal. (1988) Nature 331:624-627); ICAM-2, LFA-1, LFA-3 (Wallner et al.(1987) J. Exp. Med. 166:923-932); MHC class I molecules, MHC class IImolecules, B7.1-β2-microglobulin (Parnes et al. (1981) Proc. Natl. Acad.Sci. USA 78:2253-2257); chaperones such as calnexin; and MHC-linkedtransporter proteins or analogs thereof (Powis et al. (1991) Nature354:528-531). Immunomodulatory factors may also be agonists,antagonists, or ligands for these molecules. For example, soluble formsof receptors can often behave as antagonists for these types of factors,as can mutated forms of the factors themselves.

Nucleic acid molecules that encode the above-described substances, aswell as other nucleic acid molecules that are advantageous for usewithin the present invention, may be readily obtained from a variety ofsources, including, for example, depositories such as the American TypeCulture Collection, or from commercial sources such as BritishBio-Technology Limited (Cowley, Oxford England). Representative examplesinclude BBG 12 (containing the GM-CSF gene coding for the mature proteinof 127 amino acids), BBG 6 (which contains sequences encoding gammainterferon), A.T.C.C. Deposit No. 39656 (which contains sequencesencoding TNF), A.T.C.C. Deposit No. 20663 (which contains sequencesencoding alpha-interferon), A.T.C.C. Deposit Nos. 31902, 31902 and 39517(which contain sequences encoding beta-interferon), A.T.C.C. Deposit No.67024 (which contains a sequence which encodes Interleukin-1b), A.T.C.C.Deposit Nos. 39405, 39452, 39516, 39626 and 39673 (which containsequences encoding Interleukin-2), A.T.C.C. Deposit Nos. 59399, 59398,and 67326 (which contain sequences encoding Interleukin-3), A.T.C.C.Deposit No. 57592 (which contains sequences encoding Interleukin-4),A.T.C.C. Deposit Nos. 59394 and 59395 (which contain sequences encodingInterleukin-5), and A.T.C.C. Deposit No. 67153 (which contains sequencesencoding Interleukin-6).

Plasmids encoding one or more of the above-identified polypeptides canbe digested with appropriate restriction enzymes, and DNA fragmentscontaining the particular gene of interest can be inserted into a genetransfer vector (e.g., expression vector as described above) usingstandard molecular biology techniques. (See, e.g., Sambrook et al.,supra, or Ausubel et al. (eds) Current Protocols in Molecular Biology,Greene Publishing and Wiley-Interscience).

Administration

The VLPs and compositions comprising these VLPs can be administered to asubject by any mode of delivery, including, for example, by parenteralinjection (e.g. subcutaneously, intraperitoneally, intravenously,intramuscularly, or to the interstitial space of a tissue), or byrectal, oral (e.g. tablet, spray), vaginal, topical, transdermal (e.g.see WO99/27961) or transcutaneous (e.g. see WO02/074244 andWO02/064162), intranasal (e.g. see WO03/028760), ocular, aural,pulmonary or other mucosal administration and/or inhalation of powdercompositions. Multiple doses can be administered by the same ordifferent routes. In a preferred embodiment, the doses are intranasallyadministered.

The VLPs (and VLP-containing compositions) can be administered prior to,concurrent with, or subsequent to delivery of other vaccines. Also, thesite of VLP administration may be the same or different as other vaccinecompositions that are being administered.

Dosage treatment with the VLP composition may be a single dose scheduleor a multiple dose schedule. A multiple dose schedule is one in which aprimary course of vaccination may be with 1-10 separate doses, followedby other doses given at subsequent time intervals, chosen to maintainand/or reinforce the immune response, for example at 1-4 months for asecond dose, and if needed, a subsequent dose(s) after several months.The dosage regimen will also, at least in part, be determined by thepotency of the modality, the vaccine delivery employed, the need of thesubject and be dependent on the judgment of the practitioner.

All patents, patent applications and publications mentioned herein arehereby incorporated by reference in their entireties.

Although disclosure has been provided in some detail by way ofillustration and example for the purposes of clarity and understanding,it will be apparent to those of skill in the art that various changesand modifications can be practiced without departing from the spirit orscope of the disclosure. Accordingly, the foregoing disclosure andfollowing examples should not be construed as limiting. Thus, it will beapparent that while exemplary results are presented with respect todengue virus, the teachings herein are equally applicable to anyflavivirus (e.g. Zika, yellow fever, Japanese encephalitis, hepatitis C,West Nile, tick-borne encephalitis) or alphavirus (e.g. chikungunya).

EXAMPLES Example 1: Dengue VLP Production

A dengue vaccine capable of rapidly eliciting a robust and balancedimmunity against the four virus serotypes after only a few immunizationsis greatly needed. We describe a new strategy to develop dengue vaccinesbased on the assembly of virus-like particles (VLPs) utilizing thestructural proteins CprME together with a modified complex of theNS2B/NS3 protease, which enhances particle formation and yield. TheseVLPs are produced in mammalian cells and resemble native dengue virus asdemonstrated by negative staining and immunogold labeling electronmicroscopy (EM). Realizing that in the mosquito that the virusreplicates at a lower temperate than in humans, we found that VLPsproduced at a lower temperature (31° C.) were recognized byconformational monoclonal antibodies (MAbs) 4G2 and 3H5, whereas VLPsproduced at a higher temperature (37° C.) were not recognized by eitherMAbs. To evaluate the significance of these conformational discrepanciesin vaccine performance, we tested the immunogenicity of VLP vaccinesproduced at 31° C. or 37° C. in alternative formulations. Mice immunizedwith the VLP vaccine produced at 31° C. (TVXDO-31° C.) elicited highertiter of neutralizing antibodies as compared to those elicited byequivalent doses of the vaccine produced at 37° C. (TVXDO-37° C.) aswell as by inactivated dengue virus vaccine or the titer seen with ahuman anti-dengue-2 convalescence serum used as a reference. Our resultsdemonstrate that the conformation of the E protein displayed on the VLPvaccine plays a critical role in the induction of highly neutralizingantibodies. These findings will guide development of a tetravalentvaccine capable of eliciting a robust and balanced neutralizing responseagainst four dengue serotypes regardless of background immunity.

Dengue is a mosquito-borne viral disease that affects humans of all agesin tropical and subtropical regions around the world. Due to globalexpansion of the mosquito vectors (primarily A. aegypti, and A.albopictus) the virus has spread to more than 120 countries, infectingapproximately 390 million people annually (1). This dramatic spread ofthe vector poses a threat to almost half of the world's population withdisease outbreaks imposing a hefty public health and economic burdenupon all affected areas. Four distinct virus serotypes (DENV1-4) can betransmitted by the bite of an infected Aedes Sp. mosquito causing aninfection characterized by fever, headache, myalgia, arthralgia and,depending on the severity of the infection, may progress to Denguehemorrhagic fever/Dengue shock syndrome (DHF/DSS) (2). Primary infectiongenerates long-term protection against the homologous serotype, but ashort-lived defense against heterologous serotypes. A secondaryinfection with a different serotype can trigger an antibody-dependentenhancement (ADE) of disease that may result in potentially fatal DHFand DSS (3). Currently, there are no specific interventions to treatdengue infections and prophylactic vaccines are the best hope to controlthe disease. The need to elicit a robust and balanced neutralizingresponse against the four-dengue serotypes, in order to prevent ADE, hashindered the development of a safe and effective dengue vaccines.

Dengue virus is a positive sense single-stranded RNA virus that belongsto the Flavivirus genus within the Flaviviridae family. There are fourdistinct identified virus serotypes: DENV-1, DENV-2, DENV-3 and DENV-4.These serotypes appeared independently during endemic cycles oftransmission between humans and arthropod vectors (4). The dengue viralgenome has a single open reading frame (ORF) encoding a polyprotein thatis cleaved co- and post-translationally by cellular and viral proteasesinto three structural proteins: capsid (C), the premembrane (prM) andthe envelope (E) as well as seven non-structural proteins: NS1, NS2A/B,NS3, NS4A/B and NS5 (5). Virus replication and morphogenesis takes placeon virus-induced remodeled endoplasmic reticulum (ER) membranes leadingto the assembly and budding of complete but immature particles into theER system. (6). The spiky immature particles undergo an additional furinprotease cleavage of prM during trafficking through the ER and thetrans-Golgi network (tGN) (7) (FIG. 1A) that together, with arearrangement of the E protein, turn the virions into smooth matureparticles that are released from infected cells.

The surface E protein is the main antigenic determinant of the virus andthe target of the immune system. Immunity directed toward the E proteinis primarily mediated by neutralizing antibody which, when present,confers protection against dengue (8).

After decades of ceaseless effort, recently a dengue vaccine has reachedcommercialization stage. This live-attenuated chimeric vaccine is basedon the yellow fever virus 17D vaccine strain, which provides thebackbone to carry the DENV prM-E structural proteins of each serotype aschimeric viruses. Results of clinical phase III trials in Asia and SouthAmerica show better results than did the clinical phase II trials, butboth present serotype-dependent differences in vaccine efficacy and alower efficacy in young children. In particular, the response againstDENV-2 is suboptimal and after three immunization doses, the vaccinationefficacy is only approximately 35% to 50% (9-11). Other vaccinecandidates currently in clinical trials include live-attenuated virusvaccines, purified inactivated vaccine, a DNA vaccine, and a subunitvaccine (12, 13). The later vaccine formulation is comprised of fourrecombinant truncated E proteins and is currently the only recombinantvaccine candidate in clinical trials.

An alternative strategy for vaccine development involves the generationof viral-like particle (VLP) vaccine. These recombinant particles areself-assembling complex structures morphologically similar to wild-typevirus but are devoid of viral genetic material and are thereby unable toreplicate or cause infection. Flavivirus subviral particles or VLPs wereinitially detected in the supernatant of flavirius-infected cells (14)and subsequent studies have shown that the sole expression ofrecombinant prM and E were sufficient to drive the assembly and buddingof subviral particles (15-17). These structures provide an attractivestrategy for vaccine development because the particulate nature ofrecombinant VLPs composed of native proteins enhances antigenrecognition, presentation and immune stimulation (18-20). Furthermore,the system allows for surface protein engineering to optimize antigenconfiguration seeking enhancement of the immune response. The provenefficacy and safety of licensed human VLP based vaccines for HBV, HPVand HEV (21-24) provide strong evidence of the value of this approachfor vaccine development.

Here, we present data on the assembly of dengue virus-like particlesutilizing a unique set of viral structural and non structural (NS)proteins and production conditions in which suspension cultures ofmammalian cells render particles with distinct immunological propertieswhich, as vaccines, elicit the production of highly effectiveneutralizing antibodies. This study describes a new strategy toefficiently develop a VLP based dengue vaccine that is assembled withnative dengue protein and manufactured in a mammalian cell suspensionculture system suitable for scale up manufacturing.

A. Materials & Methods Genes and Plasmids Construct

The structural and non structural genes of DENV-2 were chemicallysynthesized by GeneArt (Life Technology) according to a specificallydesigned and codon-optimized sequence. DNA fragments were subcloned intothe plasmid vector pcDNA3.4 (Life Techhologies) utilizing the NheI/NotIrestriction enzyme sites. Specific mutations were introduced in the prMgene G88A, and in the E gene, I398L, M401A, M412L, as described by Purdyand Chang (25).

The non-structural genes of NS2B and viral protease NS3 were synthesizedas a single codon optimized unit and subcloned into plasmid vectorpcDNA3.4 via XhoI/EcoRV. The mutation L115A was introduced into thesynthesized NS3 gene.

Plasmids were amplified in MAX Efficiency® Stbl2™ Competent E. ColiCells (Life Technologies 10268-019) and purified from the bacteriautilizing an EndoFree Plasmid Maxi Kit (Qiagen).

Virus, Cells, and Antibodies

Cultures of Vero cells (ATCC® CCL-81™) were maintained in VP-SFM media(Life Technologies 11681-020) supplemented with 2 mM L-Glutamine (LifeTechnologies 25030-081), 2 mM GlutaMAX™ Supplement (Life Technologies35050-061), 1× Non-essential amino acids solution (Life Technologies11140-050), 1×ITSE (Invitria 777ITS032) and 500 ng/ml rhEGF (LifeTechnologies PHG0314).

Dengue virus: DENV-2 Th-36 (ATCC® VR-1810™) was amplified in Vero cellsfollowing virus inoculation at a low multiplicity of infection (MO:0.01). Expi293™ (Life Technologies) cells were expanded in Expi293medium (Gibco A1435101) and transfected using Expifectamine followingthe manufacturer's instructions (Life Technologies, A14635). Monoclonalantibody 4G2 is an in-house protein G purified from hybridomaD1-4G2-4-15 culture supernatant (ATCC HB-112). Mouse monoclonal antibody3H5 was acquired through BEI Resources (NR-2556). Rabbit polyclonalantibodies, anti-C (GTX124247), anti-E (GTX127277), anti-prM (GTX128093)and anti-NS2B (GTX124246) were purchased from GeneTex, CA.

These secondary antibodies anti-mouse and anti-rabbit were bothpurchased from Pierce Thermo Fisher, MA (#31430 and #31460respectively).

VLP Production and Purification

Expi293™ cells were transfected with a 1:2 ratio of pcDNA3.4-NS2b/NS3and pcDNA3.4-CprME at 37° C. and transferred after 4 h post-transfectionto incubators set at either 37° C. or 31° C. Transfected cells wereharvested 72 hs post-transfection and clarified via two successivecentrifugations. The first clarification was performed at 400×g for 10min at 4° C. followed by a second clarification at 10,000×g for 10 minat 4° C. Clarified supernatant fluid was concentrated byultracentrifugation for 2 h at 140,000×g at 4° C. The pellet wasresuspended with 1×PBSCaMg pH 7.2 (1× phosphate buffered salinesupplemented with 1 mM MgCl₂; and 1 mM CaCl₂)). VLPs were furtherpurified by ultracentrifugation through a 20-60% step sucrose gradientin TN buffer (50 mM Tris-HCl pH 7.2; 150 mM NaCl) for 4 h at 180,000×gat 4° C. using an SW40Ti rotor (Beckman Coulter, CA). The proteincontent of the collected fractions was analyzed by dot blot using adengue specific antibody. Selected fractions were combined, dialyzedovernight versus 1×PBS and concentrated by ultracentrifugation for 2 hat 140,000×g at 4° C. The pellet was then suspended in 80 μl of1×PBSCaMg and loaded onto a second sucrose gradient (20%-60%) in TNbuffer. Fractions were collected, analyzed and processed in the samefashion as in the first linear gradient above.

Dot Blot and Western Blot Assays

The cell protein content was analyzed after clarification of transfectedExpi293™ cells. The cell pellet was collected and cells were lysed withRIPA buffer (PI-89901, Pierce Thermo Fisher, MA). For Western blotting,cell lysates and concentrated culture supernatants were loaded onto a10-20% Tris-glycine SDS-PAGE gel (EC61352BOX, Life Technologies, CA).After electrophoresis separation, proteins were electro-transferred fromthe gel onto a 0.45 m nitrocellulose membrane (Life TechnologiesLC2001). For dot blot, 3 μl of sample was applied on top of a 0.45 mnitrocellulose membrane and allowed to dry for 5 min. The nitrocellulosemembranes were then treated for 1 h at room temperature with blockingsolution (5% non-fat milk 1×TBS 0.1% Tween-20) followed by overnightincubation at room temperature in primary antibody diluted in blockingsolution. Membranes were washed three times for 5 min with 1×TBS 0.1%Tween-20 and then incubated for 1.5 h in secondary antibody diluted inblocking solution. Finally, membranes were washed three times with1×TBS-0.1% Tween-20 and developed with ECL system (WP20005, LifeTechnologies, CA).

Negative Staining and Immuno-Gold Labeling Electron Microscopy

Gradient purified VLP samples were blotted onto 200-mesh carbon coatedgrid (EMS CF200-Cu) for 5 min. The grids were then washed and stainedwith 2% uranyl acetate (EMS 22400-2). Examination of VLPs by immunogoldlabeling EM was performed as follows: sample coated carbon grids wereblocked with 3% BSA in 0.1M Sodium cacodylate buffer for 5 min, followedby incubation for 20 min in primary monoclonal antibody 3H5 diluted inPBS (1:100 dilution). Grids were then washed three times with 0.1Msodium cacodylate buffer and then incubated for 20 min with secondarygoat anti-mouse antibody (1:30 dilution). After a final series of threewashes with 0.1M Sodium Cacodylate buffer, grids were stained with 2%uranyl acetate solution and examined with a JEOL-1400 electronmicroscope at the Rockefeller University Imaging Center.

Mouse Immunogenicity Study

Ten groups of 4-week old BALB/c mice were inoculated twice (day 0 andday 24) via the intramuscular (IM) route with VLP vaccines (TVXDO-31° C.or TVXDO37° C.). Groups of mice received doses of either 1 μg or 5 μg oftotal E protein content and formulated alone or admixed in a 1:1 volumewith a squalene-based oil-in-water nano-emulsion AddaVax (InvivoGen,CA). Control groups were immunized with formalin inactivated (0.05%)DENV-2 Th-36 virus at the dose of either 1 μg or 5 μg of total E proteincontent. Serum samples for immunogenicity evaluation were collected atday 39.

ELISA

ELISA assays were performed in 96-well plates coated with 100 μl/well offormaldehyde inactivated DENV-2 virus (1 g/ml of E protein content) andincubated overnight at 4° C. The plates were washed three times withPBST buffer (phosphate buffered saline plus 0.05% Tween-20) and thenblocked with 100 μl of blocking buffer (PBST plus 5% non-fat milk) forone hour at room temperature. Mouse sera were diluted following a 4-foldserial dilution in blocking buffer starting at 1:10; 50 μl of thediluted sera were incubated for 2 h at room temperature. After a set of6 washes with PBST, plates were incubated for 2 h with 50 μl ofHRP-conjugated goat anti-mouse antibody diluted 1:2,000 in blockingbuffer. Subsequently, plates were washed (6×) and developed by adding 50μl/well of ECL reagent. Light emission was measured at 425 nm using aplate reader (Synergy, H1; BioTek, VT).

Plaque Reduction Neutralization Test (PRNT)

The plaque reduction neutralization test was carried out in Vero cellsusing dengue virus serotype 2 (DENV-2) and sera from immunized miceaccording to the method described for the evaluation of vaccine efficacy(26, 27). Briefly, DENV-2 was amplified and titrated in Vero cells usingthe same plaque visualization procedure described below. Vero cells wereseeded in 24 well plates at a density of 5×104 cells per well 24 h priorto the initiation of the test. Control and tests sera were heatinactivated at 56° C. for 30 min. and then two-fold serially diluted incell culture media supplemented with penicillin and streptomycin. Anequal amount of diluted virus to form ˜60 plaques per well was added toeach serum dilution. The sera-virus mixture was incubated for 1 h at 37°C. in a 5% C02 environment. Subsequently, each dilution was applied induplicate wells of an 85% confluent monolayer of Vero cells andincubated for 1 h at 37° C. in a 5% C02 incubator.

Thereafter, the inoculum was removed and a 3 ml overlay of 2%carboxymethyl cellulose (CMC) in culture medium was added to each well.Plates were then incubated for six days at which time the CMC overlaywas removed, cells washed 1× with PBST (phosphate buffer saline plus0.05% Tween 20) and fixed with cold 80% acetone for 10 min at roomtemperature (RT). Subsequently, plates were washed 1× with PBST and thenincubated with blocking buffer (2.5% non-fat milk in PBS plus 0.5% ofTriton X-100) for 1 h in a 37° C. incubator. After one wash, the primaryantibody (MAb 4G2) diluted 1/200 in blocking buffer was applied for 2 hat RT, followed by two PBST washes and incubation for 1 h at RT with agoat anti-mouse AP conjugated secondary antibody ( 1/2000) in blockingbuffer. Finally, plates were washed twice with PBST and one time withalkaline phosphate buffer (APB: 100 mM Tris-HCl pH9.0, 150 mMNaCl, 1mMMgCl2). Viral plaques were detected by adding 100 μl per well of thealkaline phosphate (AP) substrate nitro blue tetrazolium chloride (NTB)and 5-brome-4-chlore-3-indolyl phosphate (BCIP) prepared and used asfollows: 33 μl of NTB (50 mg/ml in 70% dimethylformamide) was added to 5ml of APB, mixed well and then added 16.5 ul of BCIP (50 mg/ml in 100%dimethylformamide) and the mixture was used within 1 h. Plaques werecounted and PRNT50s were determined using the PROBIT method (28). Theneutralization power calculated is expressed as the reciprocal of thehighest serum dilution that neutralizes 50% of the virus. Humanpre-immune serum control and human anti-DENV-2 convalescent serumreferences were obtained from National Institute for BiologicalStandards and Control (NIBSC), UK.

B. Results

Processing of the structural and non-structural polyproteins andformation of dengue virus-like particles (VLPs)

To assemble and release dengue virus-like particles (VLPs), weco-transfected into suspension cultures of Expi293™ human cells two DNAplasmids, one expressing the structural protein CprME and the other thenon-structural proteins NS2B/NS3 (FIG. 4B). Expression of this proteincombination resulted in the processing of the structural proteins CprMEin an analogous fashion as occurs within the whole viral polyprotein(FIG. 4A) and therefore drove the formation and release of dengue VLPs.

The genes encoding CprME (plasmid TVXDO2) and NS2B/NS3 (plasmid TVXDO3)were de novo-synthesized and codon optimized to enhance proteinexpression in the HEK293 derived human cell line. Both the structuraland non-structural protein coding sequences were modified by truncationand substitution mutations to facilitate polyprotein processing andenhance assembly and release of the VLPs. We introduced mutations atpositions I398A, M104A and M412L within the amphipathic domain-1 of theE protein to overcome restrictions in the processing and maturation ofthe dengue polyprotein and thereby enhance VLP yield. (e.g. retentionsignal in the E protein) (25). We also mutated E88A in the furinrecognition sequence of prM protein to enhance furin cleavage at thislocation. Furthermore, the viral NS3 protease/helicase was truncatedretaining only the N-terminal protease domain, which contains the L115Amutation to enhance its catalytic activity. The remaining proteasecleavage sites within the CprME polypeptide, however, were preserved inorder to maintain a processing pattern analogous to the one occurring indengue virus infected cells (FIG. 4B).

Western blot analysis of transfected Expi293™ cell lysates showed notonly expression of both structural and non-structural proteins but alsocomplete processing of the structural polyprotein. Indeed, the capsid(12 Kda), the prM predicted size (23/8 Kda) and the E protein (64 KDa)demonstrated the appropriate molecular size, as would be expected of afully processed polyprotein (FIG. 5). The molecular weight of the NS2Bprotein is similar to the one detected in the DENV-2 cell lysate,suggesting that the self-cleavage between NS2B/NS3Pro occurs efficientlyat the corresponding cleavage site (FIG. 2D). The NS3 portion ofNS2B/NS3 could not be detected due to the lack of a suitable specificantibody. The efficient self-cleavage of NS2B/NS3Pro and the cleavage ofthe capsid protein confirm that the amino acids 1 to 183 are sufficientfor proper protease activity as described by Li et. al. (29). Ourconstruct contains the substitution mutation L115A in the NS3Pro, whichdoes not appear to interfere with proper protease activity.

To determine whether the NS2B/NS3pro have an effect on VLP production,we transfected Expi293™ cells with the TVXDO2 (CprME) plasmid alone orTVXDO2 together with TVXDO3 (NS2B/NS3pro) plasmid. The culturesupernatants were clarified twice and VLPs concentrated byultracentrifugation. Western blot analysis of concentrated supernatantof TVXDO2 alone showed that small amount of pr/M and E proteins weresecreted into the culture medium (FIG. 6). Release of the structuralproteins, however, was enhanced when the plasmid TVXDO2 expressing thestructural genes (CprME) was co-transfected with the plasmid TVXDO3 thatexpresses the modified non-structural proteins NS2/NS3pro. There was anincrease in the amount of secreted E and prM when NS2B/NS3pro wasco-expressed with CprME (FIG. 6). Furthermore, these results revealedinterdependence of cleavage between the C-anchored to C and the Canchored to prM, which is in agreement with findings in studiespreviously reported (30, 31). Thus, this protein combination offers aneffective approach for VLP production.

Dot Blot Analysis of VLPs Produced at Different Temperatures

Dengue virus is able to replicate in both mosquitoes and humans, whichillustrates the broad temperature range (ambient versus 37° C.respectively) over which the virus can complete its replication cycle.Several studies have shown that higher VLP yields can be obtained whenthe producing mammalian cell culture is incubated at a lower temperatureunderlying the significance of a slower assembly process (32, 33). Basedon this information, we chose to evaluate the effect of differenttemperatures on particle formation and surface protein structure by dotblot using specific MAbs recognizing conformation epitopes of the Eprotein. VLPs were produced at two distinct temperatures 31° C. and 37°C., purified through a linear sucrose gradient (15%-50%) and theirsedimentation profile analyzed by dot blot using an anti-E polyclonalantibody. This comparative examination showed that the migration profileof the VLPs produced at both temperatures was similar with the strongestreactivity in fractions 10 to 13, indicating that the differingtemperatures did not affect the migration profile of the assembled VLPs(FIG. 7).

To examine the structural conformation of the E protein, we utilized thedot blot method that allows for the study of dengue VLP in its nativeconformation. The monoclonal antibodies 4G2 and 3H5 that recognizestructural epitopes on the E protein were used to search forconformational differences between VLPs produced at 31° C. (TVXDO-31°C.) and 37° C. (TVXDO-37° C.). The MAb 4G2 recognizes a structuralepitope in the domain DII of E at the fusion loop comprised of aminoacids G104, G106, L107 and W231. The fusion loop of the E protein playsa key role in membrane fusion and therefore in dengue virus cell entryand replication.

Purified VLPs produced at 31° C. (fractions 10-13) demonstrated thestrongest reactivity with the MAb 4G2, whereas VLPs produced at 37° C.failed to be recognized by this MAb (FIG. 7). It was evident, therefore,that the recombinant E protein displayed on the VLPs produced at either31° C. or 37° C. was recognized by the anti-E polyclonal antibody butthat only the VLPs produced at 31° C. exhibited the epitope recognizedby 4G2 MAb, suggesting a conformational difference between these Eproteins.

To further examine the structural features of the VLP E protein, we useda second MAb (3H5) that recognizes a conformation epitope in the domainDIII of the E protein, comprised of amino acid residues K305, P384 whichare essential for binding to cellular receptors (34, 35). Dot blotanalysis of purified VLPs with the 3H5 MAb showed strong reactivity withTVXDO-31° C. but failed to recognize TVXDO-37° C. (FIG. 7). Thus,equivalent fractions of the two VLP preparations reacted strongly withthe anti-E polyclonal antibody, however only the VLPs prepared at 31° C.(TVXDO-31° C.) were recognized by both 3H5 and 4G2 (FIG. 7). TVXDO-31°C. shows closer immunological characteristics to the wild type virusthan does the TVXDO-37° C. VLP. All three tested antibodies recognizedDENV-2 control. These results support the conclusion that the E proteindisplay on VLPs produced at a lower temperature adopt structures thatbetter exhibit conformational epitopes, which are critical for theinduction of neutralizing antibodies.

Electron Microscopy (EM) Examination of DEN-2 VLPs

In view of the conformational differences due to incubation at either31° C. or 37° C., we carried out a closer examination of purified VLPsby negative staining and electron microscopy to further evaluate themorphology, shape, size and surface composition of the secretedvirus-like particles. EM examination of TVXDO-31° C. VLPs (FIG. 8A)showed that they are spherical in shape and with an approximately 50 nmdiameter, resembling structural characteristics of mature dengue virusas has been previously shown (36). Immuno-gold labeling EM of dengue-2VLPs using the conformational-epitope recognized by MAb 3H5 showedsurface reactivity and labeling only of TVXDO-31° C. VLP, confirming theassembly and release of particles composed of prM/E that exhibited theconformational epitopes recognized by these MAbs (FIG. 8B). In contrast,the TVXDO-37° C. VLPs did not react with either 4G2 or 3H5.

Immunogenicity Evaluation of VLPs in Mice by ELISA

The recombinant DENV-2 VLPs showed immunological and structuralsimilarities with DENV-2 virus when produced at 31° C. but not at 37° C.To ascertain whether these distinctions play a significant role in theimmunogenicity of the VLPs and therefore in their effectiveness asvaccine candidates, we performed immunization studies in BALB/c mice.Groups of mice (n=4) were immunized twice, two weeks apart, via theintramuscular route with either TVXDO-31° C. or TVXDO-37° C. at the doseof 1 g or 5 g of total E protein content and formulated with or withoutadjuvant. Two weeks after the booster immunization, we collected serumsamples from vaccine and control mice and assessed the antibody responseby measuring total IgG levels by ELISA and neutralizing antibody titersby plaque reduction neutralization assay. Mice immunized with 5 μg ofTVXDO-31° C. VLP vaccine with or without adjuvant demonstrated thehighest level of IgG production as compared to the equivalent dose andformulations of TVXDO-37° C. vaccine or the inactivated DENV-2 viruscontrol (FIG. 6). Similarly, the TVXDO-31° C. VLP vaccine administeredat the dose of 1 μg elicited production of higher IgG levels than theTVXDO-37° C. VLP or inactivated virus vaccine. Although the TVXDO-31° C.VLP vaccine induced the highest IgG levels, in a dose response manner,it was only the 1p g adjuvanted dose that showed a statisticallysignificant difference with the TVXDO-37° C. VLP vaccine (FIG. 9).

Immunogenicity Assessment by Plaque Reduction Neutralization Test (PRNT)

Elicitation of high titers of neutralizing antibody is paramount fordengue protection and therefore we measured the levels of neutralizingantibodies elicited by these vaccines via PRNT. The assay was performedaccording to the World Health Organization (WHO) guidelines andprotocols (26, 27). The PRNT50 of reciprocal dilutions was calculatedusing the PROBIT methods and compared with pre-immune mouse serum andreference controls (FIG. 10). Both adjuvanted vaccines, at the dose ofeither 1 μg or 5 μg elicited higher neutralizing titers than thenon-adjuvanted preparations. The 1 μg dose of TVXDO-37° C. vaccine, withand without adjuvant induced lower neutralizing antibody titers(PRNT50s: <25 and 57) than an equivalent dose of the inactivated denguevirus control (FIG. 10). However, the adjuvanted 5 μg dose of theTVXDO-37° C. elicited neutralizing titers (PRNT50: 382) that weregreater than the one induced by the 5 μg (PRNT50: 201) and 1 μg (PRNT50:158) doses of the inactivated dengue virus control.

On the other hand, the non-adjuvanted TVXDO-31° C. vaccine at the doseof 1 μg and 5 μg stimulated neutralizing antibody in the PRNT50 rangingfrom 99 to 196. However, when this vaccine was admixed with adjuvant thepotency of the neutralizing response increased 3.7-fold to a PRNT50 of371 for the 1 μg dose and greater than 5-fold to a PRNT50 of 1067 forthe 5 μg dose. The high neutralizing potency stimulated by theadjuvanted 5 μg dose of the TVXDO-31° C. is almost 3-fold greater thanthe equivalent dose of the TVXDO-37° C. as well as 5.3 fold greater thanthe inactivated dengue virus control. Furthermore, to better judge theneutralization power elicited by the different vaccine doses,formulations and controls, we performed PRNT50 assays with a standard ofhuman pre-immune and human convalescence DENV-2 sera obtained from theNational Institute for Biological Standard and Control, UK (NIBSC). ThePRNT50 of human seronegative control was similar to mouse pre-immunesera whereas the neutralizing power of the human convalescent DENV-2serum (PRNT50: 297), was slightly higher than the high dose (5 μg) ofinactivated dengue virus control (PRNT50: 201). On the other hand, theadjuvanted TVXDO-31° C. VLP vaccine at the dose of 1 μg and 5 μgelicited neutralizing antibody responses that were 1.2 fold and 3.6 foldhigher than the human convalescence sera control, PRNT50: 371 versus 297and PRNT50: 1067 versus 297 respectively. Although the TVXDO-37° C.adjuvanted 5 μg dose vaccine performed better than the inactivatedDENV-2 and the anti-DENV-2 human serum standard it did not reach theneutralizing power induced by the TVXDO-31° C. vaccine (PRNT: 382 versusPRNT: 1067). These results clearly show that the VLP vaccine produced atlower temperature, TVXDO-31° C., and formulated with adjuvant elicitsthe strongest anti-DENV-2 neutralizing antibody response, which potencycorrelated with the amount of antigen and inclusion of adjuvant.

Dengue is an expanding disease due to the increase numbers andgeographic spread of the Aedes sp. mosquito population that disseminatesthe virus. The only vaccine approved for DENV is a live attenuated virusthat requires a year of multiple immunizations to reach, in many cases,an unbalanced immunity against the four-dengue serotypes (Dengvaxia,Sanofi Pasteur's) (10, 11). Importantly, vaccine efficacy against themost disseminated DENV-2 serotype is only approximately 35% to 50%.Thus, new approaches for developing highly effective and safe denguevaccines are needed. Here we report a novel strategy that utilizes a newset of structural and non-structural dengue proteins to assemblevirus-like particles (VLPs) for dengue vaccine development. Most dengueVLPs have been assembled using the sole expression of prM and E proteins(37) and these attempts have revealed limitations in protein processingand domain restrictions, such as the presence of E retention sequences,which reduce assembly efficacy and yield. Our approach utilizes thecomplete set of structural proteins (CprME) expressed as a singlepolypeptide together with a modified NS2B/NS3 protease complex. In orderto optimize authentic protein processing, trafficking and particleassembly, we introduced specific mutations, substitutions and a deletionwithin the structural and non-structural proteins. We mutated the furinrecognition site (E88A) in prM protein to enhance cleavage and proteinprocessing as well as introduced changes in the helical domain 1 of theE protein (I398A, M401A, and M412L) to improve its amphipathicproperties and thus enhance trafficking and secretion of E. Lack offurin cleavage and retention of the E protein by its helical domains mayimpede protein transport and the assembly of mature particles.Furthermore, the C-terminal of the NS3 protein was truncated retainingonly its N-terminal protease domain that contains a mutation (L115A) toenhance its catalytic activity. The NS3 protease domain was geneticallylinked to its cofactor NS2B and expressed as a single polypeptide inorder to maintain enzymatic activity. We selected this approach ratherthan replacing C with a heterologous signal peptide for proteintranslocation into the ER, as has been reported (38, 39) because ouroptimized CPrME construct spurred significant expression of thestructural polyprotein, which was effectively processed by theco-expressed viral protease (NS2B/NS3) and cell host proteases.Co-expression of these proteins in suspension culture of mammalian cellsresulted in the assembly and release of dengue virus-like particles(VLPs) into the culture media. Their structures resemble native denguevirus in shape, size and surface antigenic composition as demonstratedby negative staining and immunogold labeling electron microscopy withVLPs produced at 31° C.

Because of the temperature range of dengue virus replication (ambient inthe mosquito vector and 37° C. in the human host) and the observationthat lower temperature increased the yield of dengue replicons (32, 33),we investigated whether the VLP production temperature enhances yield.This study revealed that lower temperature (31° C.) not only improvesVLP yield but also has a significant effect on the antigenic propertiesof the E protein displayed on the particle surface. VLPs produced at 31°C. were strongly recognized by two distinct MAbs (4G2 and 3H5) that bindthe conformational epitopes in domain II (4G2) and domain III (3H5) ofthe E protein. In contrast, VLPs produced at 37° C. were recognized byneither 4G2 nor 3H5, revealing that the targeted epitopes were notproperly folded in two different domains of the E protein. This findingsuggests that production temperature has a significant effect on thefolding and conformation of the E protein exhibited on the surface ofthe VLPs. Several studies have demonstrated the effect of differenttemperatures on dengue virus structures. Detailed cryo-electronmicroscopy analysis of dengue virus showed that mature virus produced at28° C. is smooth, however when incubated at temperatures higher than 33°C. it appears bumpy, with an increase in diameter and heterogeneity (40,41). Although it has been reported that MAbs react differently withdengue virus of distinct conformations (42, 43), the role of dissimilarconformations of E in the elicitation of neutralizing antibodies has notbeen completely elucidated.

In our vaccine studies, we have shown that VLPs produced at 31° C.elicit higher neutralizing antibody titers than those produced at 37° C.This finding indicates that the TVXDO-31° C. vaccine exhibits a betterconformation of E as was initially recognized with the MAbs 4G2 and 3H5.This structure appears to displays a larger repertoire of neutralizingsites, which is evident in the stronger neutralizing response stimulatedby this vaccine. Furthermore, the higher neutralizing activitystimulated by the TVXDO-31° C. VLP vaccine cannot be merely attributedto the sites recognized by 4G2 and 3H5 because these antibodies are nothighly neutralizing, suggesting that additional epitopes capable ofstimulating neutralizing antibodies are also exposed. Neutralizingtiters were markedly enhanced with the addition of an adjuvant and theimprovement was significantly higher with both doses (1 μg and 5 μg) ofTVXDO-31° C. (PRNTs: 371 and 1067) as compared to equivalent doses ofthe TVXDO-37° C. vaccine (PRNTs: 57 and 382). To provide context tothese PRNT results, we tested a human anti-DENV-2 convalescent serum(NIBSC/WHO) and found that it had lower neutralizing titers than thatobtained with either dose of the adjuvanted TVXDO-31° C. vaccine or evenhigher dose of the TVXDO-37° C. vaccine. The difference in neutralizingpower between the two VLP vaccines (TVXDO-31° C. and TVXDO-37° C.) ateither dose with and without adjuvant illustrates intrinsic distinctionsin the antigenic conformation of the E protein of the VLPs with betterdisplay, arrangement or frequency of epitopes in the TVXDO-31° C.vaccine. It is even possible that these conformational attributes ariseafter inoculation as the consequence of molecular breathing at thehigher temperature of the recipient host (37° C.). In addition, thedisplay of quaternary epitopes that result from adjacent domains of twosurface proteins may further explain why the VLPs produced at 31° C.elicit higher neutralizing titers, since molecular breathing may alsoimpact the quaternary structure of the VLP. Reactivity with MAb 4G2suggests better E protein folding, which correlates with quaternaryepitope display as has been reported (42, 44-46). These resultshighlight how conformational differences of the major dengue surfaceantigens affect the elicitation of neutralizing antibodies. Given thesignificance of the level of neutralizing antibody against dengue neededto achieve protection, these findings may be of great significance indengue vaccine design and development. While other dengue VLP vaccinestudies have reported efficacy, some of these preclinical tests usedsignificantly higher doses than the one used with our VLP vaccineformulations (100 μg versus 5 μg) (38) and the assays used to assessefficacy were not comparable to our PRNTs. Thus, it seems impractical tocompare the effectiveness of our TVXDO-31° C. or TVXDO-37° C. with othervaccine constructs. The convalescent human DENV-2 serum standard mayprovide the best benchmark for comparison.

In summary, this work not only describes a new strategy for VLP baseddengue vaccine development but also shows the effect of temperature onthe VLP E protein conformation and how these structural dissimilaritiesdictate the neutralizing antibody response, which is higher with VLPvaccines produced at a lower temperature. These findings hold greatpromise for the development of a VLP-based dengue vaccine.

Example 2: Zika VLP Production

Emerging viral infections are those that either newly infect the humanpopulation such as Zika or rapidly disseminate increasing thegeographical range of infection and the number of cases of disease, asis the case with dengue. One of the most effective countermeasures tofight these arthropod transmitted viral infections is prophylacticvaccination. We have developed a flavivirus VLP vaccine platformtechnology to generate vaccines against these pathogens and using it toproduce a Zika vaccine.

Zika fever disease results from an infection with Zika virus (ZIKV),which is transmitted to humans by the bite of an infected Aedes mosquito(A. aegypti, A. albopictus and polynesiensis). Zika virus was isolatedfor the first time from a Rhesus monkey in the Zika Forest in Uganda in1947 and later from humans in 1952 [2].

ZIKV has been transmitted in Africa for many years through a sylvaticcycle between mosquito vectors and nonhuman primates, with occasionalhuman infections [3]. In recent years, however, epidemics of Zika haveresulted from cycles of transmission between vectors and humansresulting in the spread of disease beyond the African continent intoFrench Polynesia and other Pacific regions [4-6]. Since 2015 a dramaticspread of ZIKV that began in Brazil is taking place in South America andthe Caribbean Islands with the occurrence of sporadic cases in travelersidentified in the USA and Europe [7].

Zika infection appears to cause a mild illness in most people infected,however, contracting the virus during pregnancy is associated with birthdefects, primarily microcephaly (defective brain development).Furthermore, an increase in cases of Guillain-Barre syndrome has beenobserved following ZIKV infection. The seriousness of these disordersimposes a tremendous burden on public health. In addition to vectortransmission, ZIKV is also likely transmitted via sexual contact [8],this fact taken together with its often asymptomatic nature makesdisease control more difficult.

Zika virus is a member of the flavivirus genus within the Flaviviridaefamily. This is a large group of enveloped viruses including dengue,yellow fever, West Nile, Japanese encephalitis and others that possess asingle stranded RNA genome of positive polarity, which serves as mRNAupon the infection of susceptible cells. The ZIKV RNA genome encodesonly one open reading frame (ORF, ˜10,272 nt) and translates into asingle polyprotein that is co- and post-translationally cleaved bycellular and virus-encoded proteases into three structural proteins (C,prM and E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3,NS4A, NS4B and NS5.) that enable virus replication [9, 10]. Flaviviursreplication and morphogenesis occurs closely associated withintracellular membranes and nascent virions are assembled andtransported through the secretory pathway and released at the cellsurface. Enveloped virions are composed of a cell-derived lipid bilayerencapsulating the C-protein wrapped viral RNA genome and studded withmultiple copies of the proteins E and M. During maturation within thesecretary pathway the precursor prM protein is cleaved by the host cellfurin protease to produce the small M protein and the fragment pr, whichis released upon virus egress from the cell. The surface displays Eprotein as the major antigenic determinant of the virus and mediatesreceptor binding and fusion during virus entry. Therefore, this proteinis a major target for vaccine development [11].

At this time there is no vaccine or specific treatment to control,combat or prevent ZIKV infection. The prevention of infection byvaccination represents a critical unmet need to control the spread andthe effects of the disease globally. Development of a ZIKV vaccine ishighly significant given the public health concerns raised by thedramatic spread of the disease, its possible effects and unclearepidemiology (birth defects, Guillain-Barre syndrome and sexualtransmission).

We implemented our flavivivirus virus-like particle (VLP) vaccineplatform technology to create immunoprotective countermeasures againstZika. VLP vaccines are produced in cell-based systems as structural andbiochemical mimics of wild-type viruses, however, VLPs lack viralgenetic material and are unable to replicate or cause infection.Therefore, vaccine inactivation is not required, better maintainingtheir antigenic epitopes and enhancing immunogenicity. The strategy tocreate flavivirus VLPs, and as an example Zika, is based on thesimultaneous expression in mammalian cells of the structural proteinsCprME together with a modified complex of the non-structural proteinNS2B/NS3 protease to maximize assembly and production.

These polypeptides suffice for the efficient self-assembly and releaseof particles into culture media. Since only sequence information ofviral genes is needed and because of the flexibility, speed and safetyof the technology, vaccines against emerging viruses such as Zika can begenerated rapidly and without risk of disseminating infectious material[12-14]. Furthermore, VLP vaccines can be manufactured via transienttransfection of mammalian cells with an expressing plasmid or by usingengineered and selected high producer stably transfected cells. Thislater approach will allow not only for the continuous vaccine productionto the desired scale but also for the storage of specific vaccineproducing cell lines that can be activated at the time, location andlevels required. In addition, this technology allows for the generationof combination vaccines via either blending distinct VLPs in a singleformulation or by assembling chimeric VLPs following the co-expressingof E proteins from different pathogens or serotypes.

Results

Transient transfection of the structural protein CprME andnon-structural protease complex NS2B/NS3 lead to secretion of particles.Purification of ZIKA VLPs by ultracentrifugation through a potassiumtartrate (10-35%)/glycerol (7%-30%) linear demonstrates a similarmigration pattern as the ZIKV when gradient fractions were probed withan anti-E specific MAb, as shown in FIG. 12. Furthermore, examination ofE positive gradient fractions of negative staining electron microscopy(EM) showed the presence of spherical particles (˜60 nm diameter) thatclosely resembled the structure of the wild type ZIKV. (FIGS. 11A, 11B,11C). To assess whether the E protein was present on the surface of theVLPs, we evaluated the same ZIKA VLP fraction by immune-gold labeling EMusing an anti-E MAb as primary and an anti-mouse gold bead conjugatedsecondary antibodies, respectively.

This study demonstrated that indeed the E protein was detected on theVLPs as shown by the presence of beads on the particles surface (FIGS.11, 11D, 11E, and 11F). In addition, Western blot analysis of ZIKA VLPsand wild type Zika virus revealed the presence of a correct size Eprotein in both VLP and virus samples when probed with anti-E antibody,FIG. 13. These data demonstrates that transfected cells release VLPs,which resemble native Zika virus and display the E protein a majorantigenic target for the elicitation of neutralizing antibodies.

The immunogenic attributes of alternative ZIKA VLP vaccine formulationsare assessed in mice to evaluate the quality and magnitude of ZIKA virusspecific neutralizing antibody response.

Based on the high neutralizing antibody titers elicited by a monovalentdengue-2 VLP vaccine, assessed by plaque reduction neutralization assay(PRNT) (FIG. 10), a ZIKA VLP vaccine should also stimulate a robustvirus neutralizing antibody response. Vaccine for flaviviruses based onthe VLP platform technology offers a safe, effective and scalable systemand warrants further development, particularly for the Zika virusproblem.

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SEQUENCES

Exemplary sequences are shown below. It will be apparent that the sameproteins can be used from any flavivirus, for example by aligning thesequences to those disclosed herein. The particular amino acid residuesidentified herein are numbered relative to the indicated sequences buttheir relative numbering in other flavivirus proteins can be readilydetermined, for example by alignment.

Dengue virus 2 Strain 168861 (GenBank U87411.1)1. DEN-2 CprME wild-type nucleotide sequence (SEQ ID NO: 1)ATGAATAACCAACGGAAAAAGGCGAAAAACACGCCTTTCAATATGCTGAAACGCGAGAGAAACCGCGTGTCGACTGTGCAACAGCTGACAAAGAGATTCTCACTTGGAATGCTGCAGGGACGAGGACCATTAAAACTGTTCATGGCCCTGGTGGCGTTCCTTCGTTTCCTAACAATCCCACCAACAGCAGGGATATTGAAGAGATGGGGAACAATTAAAAAATCAAAAGCTATTAATGTTTTGAGAGGGTTCAGGAAAGAGATTGGAAGGATGCTGAACATCTTGAATAGGAGACGCAGATCTGCAGGCATGATCATTATGCTGATTCCAACAGTGATGGCGTTCCATTTAACCACACGTAACGGAGAACCACACATGATCGTCAGCAGACAAGAGAAAGGGAAAAGTCTTCTGTTTAAAACAGAGGATGGCGTGAACATGTGTACCCTCATGGCCATGGACCTTGGTGAATTGTGTGAAGACACAATCACGTACAAGTGTCCCCTTCTCAGGCAGAATGAGCCAGAAGACATAGACTGTTGGTGCAACTCTACGTCCACGTGGGTAACTTATGGGACGTGTACCACCATGGGAGAACATAGAAGAGAAAAAAGATCAGTGGCACTCGTTCCACATGTGGGAATGGGACTGGAGACACGAACTGAAACATGGATGTCATCAGAAGGGGCCTGGAAACATGTCCAGAGAATTGAAACTTGGATCTTGAGACATCCAGGCTTCACCATGATGGCAGCAATCCTGGCATACACCATAGGAACGACACATTTCCAAAGAGCCCTGATTTTCATCTTACTGACAGCTGTCACTCCTTCAATGACAATGCGTTGCATAGGAATGTCAAATAGAGACTTTGTGGAAGGGGTTTCAGGAGGAAGCTGGGTTGACATAGTCTTAGAACATGGAAGCTGTGTGACGACGATGGCAAAAAACAAACCAACATTGGATTTTGAACTGATAAAAACAGAAGCCAAACAGCCTGCCACCCTAAGGAAGTACTGTATAGAGGCAAAGCTAACCAACACAACAACAGAATCTCGCTGCCCAACACAAGGGGAACCCAGCCTAAATGAAGAGCAGGACAAAAGGTTCGTCTGCAAACACTCCATGGTAGACAGAGGATGGGGAAATGGATGTGGACTATTTGGAAAGGGAGGCATTGTGACCTGTGCTATGTTCAGATGCAAAAAGAACATGGAAGGAAAAGTTGTGCAACCAGAAAACTTGGAATACACCATTGTGATAACACCTCACTCAGGGGAAGAGCATGCAGTCGGAAATGACACAGGAAAACATGGCAAGGAAATCAAAATAACACCACAGAGTTCCATCACAGAAGCAGAATTGACAGGTTATGGCACTGTCACAATGGAGTGCTCTCCAAGAACGGGCCTCGACTTCAATGAGATGGTGTTGCTGCAGATGGAAAATAAAGCTTGGCTGGTGCACAGGCAATGGTTCCTAGACCTGCCGTTACCATGGTTGCCCGGAGCGGACACACAAGGGTCAAATTGGATACAGAAAGAGACATTGGTCACTTTCAAAAATCCCCATGCGAAGAAACAGGATGTTGTTGTTTTAGGATCCCAAGAAGGGGCCATGCACACAGCACTTACAGGGGCCACAGAAATCCAAATGTCATCAGGAAACTTACTCTTCACAGGACATCTCAAGTGCAGGCTGAGAATGGACAAGCTACAGCTCAAAGGAATGTCATACTCTATGTGCACAGGAAAGTTTAAAGTTGTGAAGGAAATAGCAGAAACACAACATGGAACAATAGTTATCAGAGTGCAATATGAAGGGGACGGCTCTCCATGCAAGATCCCTTTTGAGATAATGGATTTGGAAAAAAGACATGTCTTAGGTCGCCTGATTACAGTCAACCCAATTGTGACAGAAAAAGATAGCCCAGTCAACATAGAAGCAGAACCTCCATTCGGAGACAGCTACATCATCATAGGAGTAGAGCCGGGACAACTGAAGCTCAACTGGTTTAAGAAAGGAAGTTCTATCGGCCAAATGTTTGAGACAACAATGAGGGGGGCGAAGAGAATGGCCATTTTAGGTGACACAGCCTGGGATTTTGGATCCTTGGGAGGAGTGTTTACATCTATAGGAAAGGCTCTCCACCAAGTCTTTGGAGCAATCTATGGAGCTGCCTTCAGTGGGGTTTCATGGACTATGAAAATCCTCATAGGAGTCATTATCACATGGATAGGAATGAATTCACGCAGCACCTCACTGTCTGTGACACTAGTATTGGTGGGAATTGTGACACTGTATTTGGGAGTCATGGTGCAGGCC2. DEN-2 CprME wild-type amino acid sequence (SEQ ID NO: 2)MNNQRKKAKNTPFNMLKRERNRVSTVQQLTKRFSLGMLQGRGPLKLFMALVAFLRFLTIPPTAGILKRWGTIKKSKAINVLRGFRKEIGRMLNILNRRRRSAGMIIMLIPTVMAFHLTTRNGEPHMIVSRQEKGKSLLFKTEDGVNMCTLMAMDLGELCEDTITYKCPLLRQNEPEDIDCWCNSTSTWVTYGTCTTMGEHRREKRSVALVPHVGMGLETRTETWMSSEGAWKHVQRIETWILRHPGFTMMAAILAYTIGTTHFQRALIFILLTAVTPSMTMRCIGMSNRDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEAKQPATLRKYCIEAKLTNTTTESRCPTQGEPSLNEEQDKRFVCKHSMVDRGWGNGCGLFGKGGIVTCAMFRCKKNMEGKVVQPENLEYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSITEAELTGYGTVTMECSPRTGLDFNEMVLLQMENKAWLVHRQWFLDLPLPWLPGADTQGSNWIQKETLVTFKNPHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFKVVKEIAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLNWFKKGSSIGQMFETTMRGAKRMAILGDTAWDFGSLGGVFTSIGKALHQVFGAIYGAAFSGVSWTMKILIGVIITWIGMNSRSTSLSVTLVLVGIV TLYLGVMVQA3. DEN-2 CprME wild-type nucleotide sequence with Mutations(SEQ ID NO: 3) ATGAATAACCAACGGAAAAAGGCGAAAAACACGCCTTTCAATATGCTGAAACGCGAGAGAAACCGCGTGTCGACTGTGCAACAGCTGACAAAGAGATTCTCACTTGGAATGCTGCAGGGACGAGGACCATTAAAACTGTTCATGGCCCTGGTGGCGTTCCTTCGTTTCCTAACAATCCCACCAACAGCAGGGATATTGAAGAGATGGGGAACAATTAAAAAATCAAAAGCTATTAATGTTTTGAGAGGGTTCAGGAAAGAGATTGGAAGGATGCTGAACATCTTGAATAGGAGACGCAGATCTGCAGGCATGATCATTATGCTGATTCCAACAGTGATGGCGTTCCATTTAACCACACGTAACGGAGAACCACACATGATCGTCAGCAGACAAGAGAAAGGGAAAAGTCTTCTGTTTAAAACAGAGGATGGCGTGAACATGTGTACCCTCATGGCCATGGACCTTGGTGAATTGTGTGAAGACACAATCACGTACAAGTGTCCCCTTCTCAGGCAGAATGAGCCAGAAGACATAGACTGTTGGTGCAACTCTACGTCCACGTGGGTAACTTATGGGACGTGTACCACCATGGGAGAACATAGAAGAGCAAAAAGATCAGTGGCACTCGTTCCACATGTGGGAATGGGACTGGAGACACGAACTGAAACATGGATGTCATCAGAAGGGGCCTGGAAACATGTCCAGAGAATTGAAACTTGGATCTTGAGACATCCAGGCTTCACCATGATGGCAGCAATCCTGGCATACACCATAGGAACGACACATTTCCAAAGAGCCCTGATTTTCATCTTACTGACAGCTGTCACTCCTTCAATGACAATGCGTTGCATAGGAATGTCAAATAGAGACTTTGTGGAAGGGGTTTCAGGAGGAAGCTGGGTTGACATAGTCTTAGAACATGGAAGCTGTGTGACGACGATGGCAAAAAACAAACCAACATTGGATTTTGAACTGATAAAAACAGAAGCCAAACAGCCTGCCACCCTAAGGAAGTACTGTATAGAGGCAAAGCTAACCAACACAACAACAGAATCTCGCTGCCCAACACAAGGGGAACCCAGCCTAAATGAAGAGCAGGACAAAAGGTTCGTCTGCAAACACTCCATGGTAGACAGAGGATGGGGAAATGGATGTGGACTATTTGGAAAGGGAGGCATTGTGACCTGTGCTATGTTCAGATGCAAAAAGAACATGGAAGGAAAAGTTGTGCAACCAGAAAACTTGGAATACACCATTGTGATAACACCTCACTCAGGGGAAGAGCATGCAGTCGGAAATGACACAGGAAAACATGGCAAGGAAATCAAAATAACACCACAGAGTTCCATCACAGAAGCAGAATTGACAGGTTATGGCACTGTCACAATGGAGTGCTCTCCAAGAACGGGCCTCGACTTCAATGAGATGGTGTTGCTGCAGATGGAAAATAAAGCTTGGCTGGTGCACAGGCAATGGTTCCTAGACCTGCCGTTACCATGGTTGCCCGGAGCGGACACACAAGGGTCAAATTGGATACAGAAAGAGACATTGGTCACTTTCAAAAATCCCCATGCGAAGAAACAGGATGTTGTTGTTTTAGGATCCCAAGAAGGGGCCATGCACACAGCACTTACAGGGGCCACAGAAATCCAAATGTCATCAGGAAACTTACTCTTCACAGGACATCTCAAGTGCAGGCTGAGAATGGACAAGCTACAGCTCAAAGGAATGTCATACTCTATGTGCACAGGAAAGTTTAAAGTTGTGAAGGAAATAGCAGAAACACAACATGGAACAATAGTTATCAGAGTGCAATATGAAGGGGACGGCTCTCCATGCAAGATCCCTTTTGAGATAATGGATTTGGAAAAAAGACATGTCTTAGGTCGCCTGATTACAGTCAACCCAATTGTGACAGAAAAAGATAGCCCAGTCAACATAGAAGCAGAACCTCCATTCGGAGACAGCTACATCATCATAGGAGTAGAGCCGGGACAACTGAAGCTCAACTGGTTTAAGAAAGGAAGTTCTTTGGGCCAAGCCTTTGAGACAACAATGAGGGGGGCGAAGAGATTGGCCATTTTAGGTGACACAGCCTGGGATTTTGGATCCTTGGGAGGAGTGTTTACATCTATAGGAAAGGCTCTCCACCAAGTCTTTGGAGCAATCTATGGAGCTGCCTTCAGTGGGGTTTCATGGACTATGAAAATCCTCATAGGAGTCATTATCACATGGATAGGAATGAATTCACGCAGCACCTCACTGTCTGTGACACTAGTATTGGTGGGAATTGTGACACTGTATTTGGGAGTCATGGTGCAGGCCTAG4. DEN-2 CprME codon optimized nucleotide sequence with Mutations(SEQ ID NO: 4) ATGAACAACCAGCGCAAGAAGGCCAAAAACACTCCGTTCAATATGCTCAAGAGAGAGCGCAATCGGGTTTCTACGGTACAGCAGCTGACGAAGAGATTCTCCCTGGGCATGCTGCAAGGTCGCGGACCACTGAAGCTGTTCATGGCCCTTGTTGCATTTCTTAGGTTTCTTACAATTCCCCCCACTGCTGGAATCCTGAAGCGGTGGGGCACCATCAAAAAGTCCAAGGCTATTAATGTCCTCAGGGGGTTCAGGAAAGAGATTGGGCGGATGCTGAACATCCTTAATAGACGCAGACGGTCCGCTGGCATGATAATCATGCTGATCCCAACCGTCATGGCCTTCCACCTGACTACCCGAAATGGAGAGCCCCACATGATCGTGAGCAGACAAGAGAAGGGGAAGAGTCTCCTGTTCAAGACAGAAGATGGCGTGAACATGTGCACACTGATGGCCATGGATCTCGGCGAACTGTGTGAAGACACTATCACCTACAAATGCCCTCTCCTCCGCCAAAATGAACCAGAAGATATTGACTGTTGGTGTAATTCAACAAGTACATGGGTGACCTATGGCACCTGTACCACCATGGGTGAACATCGACGGGCGAAGAGAAGTGTGGCCCTCGTCCCTCATGTCGGCATGGGGTTGGAGACCAGAACAGAAACCTGGATGAGCTCCGAGGGCGCCTGGAAACACGTGCAGCGGATCGAAACATGGATTTTGCGGCACCCTGGATTTACTATGATGGCTGCCATTCTGGCTTACACAATAGGCACCACACACTTTCAGCGGGCGCTTATCTTTATCCTCCTGACAGCAGTAACACCCTCTATGACCATGAGGTGCATCGGGATGTCAAACCGGGACTTTGTTGAGGGGGTCAGCGGTGGATCTTGGGTGGATATCGTGCTGGAACACGGATCTTGCGTGACCACCATGGCAAAGAATAAGCCCACACTTGATTTTGAATTGATTAAAACCGAGGCAAAACAGCCAGCCACTCTTCGCAAATACTGCATCGAGGCCAAGCTGACCAACACAACCACAGAATCCCGATGCCCTACCCAAGGTGAGCCTTCTCTTAACGAAGAGCAGGACAAACGGTTCGTGTGTAAGCATTCCATGGTGGACAGGGGGTGGGGGAATGGATGCGGGCTCTTCGGGAAGGGCGGGATCGTCACATGTGCAATGTTTAGATGTAAGAAAAATATGGAAGGCAAAGTTGTGCAGCCAGAGAATCTGGAATATACTATTGTGATAACGCCTCACTCAGGTGAAGAGCACGCTGTAGGCAACGATACCGGCAAGCACGGAAAGGAAATCAAGATAACTCCACAGTCAAGCATCACGGAAGCTGAGTTGACAGGGTACGGCACTGTCACCATGGAGTGCAGCCCACGCACTGGTCTGGACTTCAATGAGATGGTGCTTCTCCAGATGGAAAACAAAGCCTGGCTGGTTCATAGGCAATGGTTTCTTGATCTTCCTTTGCCCTGGCTGCCTGGAGCAGATACACAGGGTTCTAATTGGATCCAAAAGGAAACCCTTGTGACCTTCAAGAACCCGCATGCAAAGAAGCAGGATGTGGTGGTCCTCGGCTCTCAGGAGGGAGCCATGCACACCGCCCTTACCGGAGCCACAGAGATCCAGATGAGCTCTGGTAACCTCCTCTTTACCGGGCATCTGAAGTGTCGCCTTAGAATGGATAAACTGCAGCTCAAAGGTATGTCCTACAGCATGTGCACAGGTAAATTCAAAGTGGTGAAGGAAATAGCTGAGACTCAGCACGGCACAATCGTCATCCGAGTTCAATATGAGGGTGACGGAAGCCCATGTAAAATCCCTTTCGAAATTATGGACCTGGAAAAACGCCACGTGCTGGGCCGACTTATCACTGTTAATCCCATAGTCACAGAGAAGGACTCTCCAGTTAACATCGAGGCCGAGCCTCCCTTCGGGGACTCCTATATCATCATCGGCGTTGAACCTGGCCAATTGAAGCTGAACTGGTTCAAAAAGGGTTCCTCACTGGGACAGGCCTTCGAAACGACAATGAGAGGCGCAAAGAGACTGGCTATCCTCGGCGATACAGCATGGGACTTCGGGTCCCTGGGAGGAGTATTCACAAGCATAGGGAAGGCCCTGCACCAAGTGTTCGGTGCGATCTATGGTGCGGCCTTCTCAGGAGTCAGTTGGACCATGAAGATTCTGATTGGCGTCATAATTACGTGGATTGGTATGAATTCAAGGTCTACATCTTTGTCCGTGACTCTGGTCCTGGTGGGAATTGTGACTTTGTATCTCGGCGTGATGGTGCAAGCCTAG 5. DEN-2 CPrME amino acid sequence with mutations(SEQ ID NO: 5) MNNQRKKAKNTPFNMLKRERNRVSTVQQLTKRFSLGMLQGRGPLKLFMALVAFLRFLTIPPTAGILKRWGTIKKSKAINVLRGFRKEIGRMLNILNRRRRSAGMIIMLIPTVMAFHLTTRNGEPHMIVSRQEKGKSLLFKTEDGVNMCTLMAMDLGELCEDTITYKCPLLRQNEPEDIDCWCNSTSTWVTYGTCTTMGEHRRAKRSVALVPHVGMGLETRTETWMSSEGAWKHVQRIETWILRHPGFTMMAAILAYTIGTTHFQRALIFILLTAVTPSMTMRCIGMSNRDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEAKQPATLRKYCIEAKLTNTTTESRCPTQGEPSLNEEQDKRFVCKHSMVDRGWGNGCGLFGKGGIVTCAMFRCKKNMEGKVVQPENLEYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSITEAELTGYGTVTMECSPRTGLDFNEMVLLQMENKAWLVHRQWFLDLPLPWLPGADTQGSNWIQKETLVTFKNPHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFKVVKEIAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLNWFKKGSSLGQAFETTMRGAKRLAILGDTAWDFGSLGGVFTSIGKALHQVFGAIYGAAFSGVSWTMKILIGVIITWIGMNSRSTSLSVTLVLVGIV TLYLGVMVQA6. DEN-2 NS2BNS3 wild-type nucleotide sequence (SEQ ID NO: 6)AGCTGGCCATTAAATGAGGCTATCATGGCAGTCGGGATGGTGAGCATTTTAGCCAGTTCTCTCCTAAAAAATGATATTCCCATGACAGGACCATTAGTGGCTGGAGGGCTCCTCACTGTGTGCTACGTGCTCACTGGACGATCGGCCGATTTGGAACTGGAGAGAGCAGCCGATGTCAAATGGGAAGACCAGGCAGAGATATCAGGAAGCAGTCCAATCCTGTCAATAACAATATCAGAAGATGGTAGCATGTCGATAAAAAATGAAGAGGAAGAACAAACACTGACCATACTCATTAGAACAGGATTGCTGGTGATCTCAGGACTTTTTCCTGTATCAATACCAATCACGGCAGCAGCATGGTACCTGTGGGAAGTGAAGAAACAACGGGCCGGAGTATTGTGGGATGTTCCTTCACCCCCACCCATGGGAAAGGCTGAACTGGAAGATGGAGCCTATAGAATTAAGCAAAAAGGGATTCTTGGATATTCCCAGATCGGAGCCGGAGTTTACAAAGAAGGAACATTCCATACAATGTGGCATGTCACACGTGGCGCTGTTCTAATGCATAAAGGAAAGAGGATTGAACCATCATGGGCGGACGTCAAGAAAGACCTAATATCATATGGAGGAGGCTGGAAGTTAGAAGGAGAATGGAAGGAAGGAGAAGAAGTCCAGGTATTGGCACTGGAGCCTGGAAAAAATCCAAGAGCCGTCCAAACGAAACCTGGTCTTTTCAAAACCAACGCCGGAACAATAGGTGCTGTATCTCTGGACTTTTCTCCTGGAACGTCAGGATCTCCAATTATCGACAAAAAAGGAAAAGTTGTGGGTCTTTATGGTAATGGTGTTGTTACAAGGAGTGGAGCATATGTGAGTGCTATAGCCCAGACTGAAAAAAGCATTGAAGACAACCCAGAGATCGAAGATGACATTTTCCGAAAGAGAAGACTGACCATCATGGACCTCCACCCAGGAGCGGGAAAGACGAAGAGATACCTTCCGGCCATAGTCAGAGAAGCTATAAAACGGGGTTTGAGAACATTAATCTTGGCCCCCACTAGAGTTGTGGCAGCTGAAATGGAGGAAGCCCTTAGAGGACTTCCAATAAGATACCAGACCCCAGCCATCAGAGCTGAGCACACCGGGCGGGAGATTGTGGACCTAATGTGTCATGCCACATTTACCATGAGGCTGCTATCACCAGTTAGAGTGCCAAACTACAACCTGATTATCATGGACGAAGCCCATTTCACAGACCCAGCAAGTATAGCAGCTAGAGGATACATCTCAACTCGAGTGGAGATGGGTGAGGCAGCTGGGATTTTTATGACAGCCACTCCCCCGGGAAGCAGAGACCCATTTCCTCAGAGCAATGCACCAATCATAGATGAAGAAAGAGAAATCCCTGAACGTTCGTGGAATTCCGGACATGAATGGGTCACGGATTTTAAAGGGAAGACTGTTTGGTTCGTTCCAAGTATAAAAGCAGGAAATGATATAGCAGCTTGCCTGAGGAAAAATGGAAAGAAAGTGATACAACTCAGTAGGAAGACCTTTGATTCTGAGTATGTCAAGACTAGAACCAATGATTGGGACTTCGTGGTTACAACTGACATTTCAGAAATGGGTGCCAATTTCAAGGCTGAGAGGGTTATAGACCCCAGACGCTGCATGAAACCAGTCATACTAACAGATGGTGAAGAGCGGGTGATTCTGGCAGGACCTATGCCAGTGACCCACTCTAGTGCAGCACAAAGAAGAGGGAGAATAGGAAGAAATCCAAAAAATGAGAATGACCAGTACATATACATGGGGGAACCTCTGGAAAATGATGAAGACTGTGCACACTGGAAAGAAGCTAAAATGCTCCTAGATAACATCAACACGCCAGAAGGAATCATTCCTAGCATGTTCGAACCAGAGCGTGAAAAGGTGGATGCCATTGATGGCGAATACCGCTTGAGAGGAGAAGCAAGGAAAACCTTTGTAGACTTAATGAGAAGAGGAGACCTACCAGTCTGGTTGGCCTACAGAGTGGCAGCTGAAGGCATCAACTACGCAGACAGAAGGTGGTGTTTTGATGGAGTCAAGAACAACCAAATCCTAGAAGAAAACGTGGAAGTTGAAATCTGGACAAAAGAAGGGGAAAGGAAGAAATTGAAACCCAGATGGTTGGATGCTAGGATCTATTCTGACCCACTGGCGCTAAAAGAATTTAAGGAATTTGCAGCCGGAAGAAAG7. DEN-2 NS2B/NS3 wild-type amino acid sequence (SEQ ID NO: 7)MSWPLNEAIMAVGMVSILASSLLKNDIPMTGPLVAGGLLTVCYVLTGRSADLELERAADVKWEDQAEISGSSPILSITISEDGSMSIKNEEEEQTLTILIRTGLLVISGLFPVSIPITAAAWYLWEVKKQRAGVLWDVPSPPPMGKAELEDGAYRIKQKGILGYSQIGAGVYKEGTFHTMWHVTRGAVLMHKGKRIEPSWADVKKDLISYGGGWKLEGEWKEGEEVQVLALEPGKNPRAVQTKPGLFKTNAGTIGAVSLDFSPGTSGSPIIDKKGKVVGLYGNGVVTRSGAYVSAIAQTEKSIEDNPEIEDDIFRKRRLTIMDLHPGAGKTKRYLPAIVREAIKRGLRTLILAPTRVVAAEMEEALRGLPIRYQTPAIRAEHTGREIVDLMCHATFTMRLLSPVRVPNYNLIIMDEAHFTDPASIAARGYISTRVEMGEAAGIFMTATPPGSRDPFPQSNAPIIDEEREIPERSWNSGHEWVTDFKGKTVWFVPSIKAGNDIAACLRKNGKKVIQLSRKTFDSEYVKTRTNDWDFVVTTDISEMGANFKAERVIDPRRCMKPVILTDGEERVILAGPMPVTHSSAAQRRGRIGRNPKNENDQYIYMGEPLENDEDCAHWKEAKMLLDNINTPEGIIPSMFEPEREKVDAIDGEYRLRGEARKTFVDLMRRGDLPVWLAYRVAAEGINYADRRWCFDGVKNNQILEENVEVEIWTKEGERKKLKPRWLDARIYSDPLALKEFKEFAAGR8. DEN-2 NS2B/NS3Pro codon optimization nucleotidesequence with mutations (SEQ ID NO: 8)ATGAGTTGGCCTCTGAACGAGGCAATAATGGCGGTGGGGATGGTGAGCATACTTGCATCAAGCCTGCTGAAGAACGACATCCCTATGACTGGTCCCCTGGTGGCCGGCGGCCTGCTGACGGTCTGTTATGTGCTGACCGGCAGGTCCGCAGACTTGGAGCTGGAAAGGGCTGCCGACGTCAAGTGGGAGGACCAGGCCGAAATTTCAGGAAGCAGTCCCATCCTGAGTATCACAATTTCCGAGGACGGTTCAATGTCCATCAAGAATGAAGAGGAAGAGCAGACACTGACCATACTGATTCGCACCGGCCTGCTTGTTATTAGTGGCTTGTTCCCTGTATCTATCCCTATCACTGCCGCCGCCTGGTATCTCTGGGAAGTAAAGAAGCAGCGGGCAGGCGTACTCTGGGATGTGCCTTCCCCCCCACCTATGGGAAAGGCGGAACTGGAGGACGGTGCATACCGCATTAAGCAAAAAGGCATCCTCGGATACAGCCAGATCGGAGCCGGGGTGTATAAAGAAGGAACCTTTCATACTATGTGGCACGTGACTAGAGGGGCGGTGCTTATGCATAAGGGTAAAAGGATTGAACCATCCTGGGCAGATGTGAAAAAAGACCTGATCTCCTACGGGGGTGGCTGGAAGCTGGAAGGGGAATGGAAGGAAGGAGAGGAGGTTCAGGTCCTTGCCCTGGAACCAGGTAAGAATCCCCGCGCCGTGCAGACAAAGCCGGGCGCTTTCAAGACTAATGCGGGAACCATCGGAGCTGTCTCCTTGGATTTCAGCCCAGGCACCTCCGGTTCTCCCATCATCGACAAAAAGGGCAAAGTCGTTGGGCTCTATGGGAATGGGGTGGTGACGCGCTCAGGAGCCTATGTTTCTGCAATAGCTCAGACCGAGAAGTCAATCGAAGATAACCCGGAAATCGAAGATGATTAG9. DEN-2 NS2B/NS3Pro amino acid sequence with mutations (SEQ ID NO: 9)MSWPLNEAIMAVGMVSILASSLLKNDIPMTGPLVAGGLLTVCYVLTGRSADLELERAADVKWEDQAEISGSSPILSITISEDGSMSIKNEEEEQTLTILIRTGLLVISGLFPVSIPITAAAWYLWEVKKQRAGVLWDVPSPPPMGKAELEDGAYRIKQKGILGYSQIGAGVYKEGTFHTMWHVTRGAVLMHKGKRIEPSWADVKKDLISYGGGWKLEGEWKEGEEVQVLALEPGKNPRAVQTKPGAFKTNAGTIGAVSLDFSPGTSGSPIIDKKGKVVGLYGNGVVTRSGAYVSAIAQTEKSIEDN PEIEDD10. DEN-2 NS2B codon optimized nucleotide sequence (SEQ ID NO: 10)ATGAGTTGGCCTCTGAACGAGGCAATAATGGCGGTGGGGATGGTGAGCATACTTGCATCAAGCCTGCTGAAGAACGACATCCCTATGACTGGTCCCCTGGTGGCCGGCGGCCTGCTGACGGTCTGTTATGTGCTGACCGGCAGGTCCGCAGACTTGGAGCTGGAAAGGGCTGCCGACGTCAAGTGGGAGGACCAGGCCGAAATTTCAGGAAGCAGTCCCATCCTGAGTATCACAATTTCCGAGGACGGTTCAATGTCCATCAAGAATGAAGAGGAAGAGCAGACACTGACCATACTGATTCGCACCGGCCTGCTTGTTATTAGTGGCTTGTTCCCTGTATCTATCCCTATCACTGCCGCCGCCTGGTATCTCTGGGAAGTAAAGAAGCAGCGGTAG11. DEN-2 NS2B amino acid sequence (SEQ ID NO: 11)MSWPLNEAIMAVGMVSILASSLLKNDIPMTGPLVAGGLLTVCYVLTGRSADLELERAADVKWEDQAEISGSSPILSITISEDGSMSIKNEEEEQTLTILIRTGLLVISGLFPVSIPITAAAWYLWEVKKQR12. DEN-2 NS3Pro codon optimized nucleotide sequence with mutation(SEQ ID NO: 12) GCAGGCGTACTCTGGGATGTGCCTTCCCCCCCACCTATGGGAAAGGCGGAACTGGAGGACGGTGCATACCGCATTAAGCAAAAAGGCATCCTCGGATACAGCCAGATCGGAGCCGGGGTGTATAAAGAAGGAACCTTTCATACTATGTGGCACGTGACTAGAGGGGCGGTGCTTATGCATAAGGGTAAAAGGATTGAACCATCCTGGGCAGATGTGAAAAAAGACCTGATCTCCTACGGGGGTGGCTGGAAGCTGGAAGGGGAATGGAAGGAAGGAGAGGAGGTTCAGGTCCTTGCCCTGGAACCAGGTAAGAATCCCCGCGCCGTGCAGACAAAGCCGGGCGCTTTCAAGACTAATGCGGGAACCATCGGAGCTGTCTCCTTGGATTTCAGCCCAGGCACCTCCGGTTCTCCCATCATCGACAAAAAGGGCAAAGTCGTTGGGCTCTATGGGAATGGGGTGGTGACGCGCTCAGGAGCCTATGTTTCTGCAATAGCTCAGACCGAGAAGTCAATCGAAGATAACCCGGAAATCGAAGATGATTAG13. DEN-2 NS3Pro amino acid sequence with mutation (SEQ ID NO: 13)AGVLWDVPSPPPMGKAELEDGAYRIKQKGILGYSQIGAGVYKEGTFHTMWHVTRGAVLMHKGKRIEPSWADVKKDLISYGGGWKLEGEWKEGEEVQVLALEPGKNPRAVQTKPGAFKTNAGTIGAVSLDFSPGTSGSPIIDKKGKVVGLYGNGVVTRSGAYVSAIAQTEKSIEDNPEIEDD 14. DEN-2 NS1 wild-type nucleotide sequence(SEQ ID NO: 14) GATAGTGGTTGCGTTGTGAGCTGGAAAAACAAAGAACTGAAATGTGGCAGTGGGATTTTCATCACAGACAACGTGCACACATGGACAGAACAATACAAGTTCCAACCAGAATCCCCTTCAAAACTAGCTTCAGCTATCCAGAAAGCCCATGAAGAGGGCATTTGTGGAATCCGCTCAGTAACAAGACTGGAGAATCTGATGTGGAAACAAATAACACCAGAATTGAATCACATTCTATCAGAAAATGAGGTGAAGTTAACTATTATGACAGGAGACATCAAAGGAATCATGCAGGCAGGAAAACGATCTCTGCGGCCTCAGCCCACTGAGCTGAAGTATTCATGGAAAACATGGGGCAAAGCAAAAATGCTCTCTACAGAGTCTCATAACCAGACCTTTCTCATTGATGGCCCCGAAACAGCAGAATGCCCCAACACAAATAGAGCTTGGAATTCGTTGGAAGTTGAAGACTATGGCTTTGGAGTATTCACCACCAATATATGGCTAAAATTGAAAGAAAAACAGGATGTATTCTGCGACTCAAAACTCATGTCAGCGGCCATAAAAGACAACAGAGCCGTCCATGCCGATATGGGTTATTGGATAGAAAGTGCACTCAATGACACATGGAAGATAGAGAAAGCCTCTTTCATTGAAGTTAAAAACTGCCACTGGCCAAAATCACACACCCTCTGGAGCAATGGAGTGCTAGAAAGTGAGATGATAATTCCAAAGAATCTCGCTGGACCAGTGTCTCAACACAACTATAGACCAGGCTACCATACACAAATAACAGGACCATGGCATCTAGGTAAGCTTGAGATGGACTTTGATTTCTGTGATGGAACAACAGTGGTAGTGACTGAGGACTGCGGAAATAGAGGACCCTCTTTGAGAACAACCACTGCCTCTGGAAAACTCATAACAGAATGGTGCTGCCGATCTTGCACATTACCACCGCTAAGATACAGAGGTGAGGATGGGTGCTGGTACGGGATGGAAATCAGACCATTGAAGGAGAAAGAAGAGAATTTGGTCAACTCCTTGGTCA CAGCTGGACATGGGCAGG15. DEN-2 NS1 wild-type amino acid sequence (SEQ ID NO: 15)DSGCVVSWKNKELKCGSGIFITDNVHTWTEQYKFQPESPSKLASAIQKAHEEGICGIRSVTRLENLMWKQITPELNHILSENEVKLTIMTGDIKGIMQAGKRSLRPQPTELKYSWKTWGKAKMLSTESHNQTFLIDGPETAECPNTNRAWNSLEVEDYGFGVFTTNIWLKLKEKQDVFCDSKLMSAAIKDNRAVHADMGYWIESALNDTWKIEKASFIEVKNCHWPKSHTLWSNGVLESEMIIPKNLAGPVSQHNYRPGYHTQITGPWHLGKLEMDFDFCDGTTVVVTEDCGNRGPSLRTTTASGKLITEWCCRSCTLPPLRYRGEDGCWYGMEIRPLKEKEENLVNSLVTAGHGQ16. DEN-2 NS2A wild-type nucleotide sequence (SEQ ID NO: 16)GACATGGGCAGGGTCGACAACTTTTCACTAGGAGTCTTGGGAATGGCATTGTTCCTGGAGGAAATGCTTAGGACCCGAGTAGGAACGAAACATGCAATACTACTAGTTGCAGTTTCTTTTGTGACATTGATCACAGGGAACATGTCCTTTAGAGACCTGGGAAGAGTGATGGTTATGGTAGGCGCCACTATGACGGATGACATAGGTATGGGCGTGACTTATCTTGCCCTACTAGCAGCCTTCAAAGTCAGACCAACTTTTGCAGCTGGACTACTCTTGAGAAAGCTGACCTCCAAGGAATTGATGATGACTACTATAGGAATTGTACTCCTCTCCCAGAGCACCATACCAGAGACCATTCTTGAGTTGACTGATGCGTTAGCCTTAGGCATGATGGTCCTCAAAATGGTGAGAAATATGGAAAAGTATCAATTGGCAGTGACTATCATGGCTATCTTGTGCGTCCCAAACGCAGTGATATTACAAAACGCATGGAAAGTGAGTTGCACAATATTGGCAGTGGTGTCCGTTTCCCCACTGCTCTTAACATCCTCACAGCAAAAAACAGATTGGATACCATTAGCATTGACGATCAAAGGTCTCAATCCAACAGCTATTTTTCTAACAACCCTCTCAAGAACCAGCAAGAAAAGG17. DEN-2 NS2A wild-type amino acid sequence (SEQ ID NO: 17)GHGQVDNFSLGVLGMALFLEEMLRTRVGTKHAILLVAVSFVTLITGNMSFRDLGRVMVMVGATMTDDIGMGVTYLALLAAFKVRPTFAAGLLLRKLTSKELMMTTIGIVLLSQSTIPETILELTDALALGMMVLKMVRNMEKYQLAVTIIVIAILCVPNAVILQNAWKVSCTILAVVSVSPLLLTSSQQKTDWIPLALTIKGLN PTAIFLTTLSRTSKKR18. DEN-2 NS3 Full length nucleotide sequence (SEQ ID NO: 18)GCCGGAGTATTGTGGGATGTTCCTTCACCCCCACCCATGGGAAAGGCTGAACTGGAAGATGGAGCCTATAGAATTAAGCAAAAAGGGATTCTTGGATATTCCCAGATCGGAGCCGGAGTTTACAAAGAAGGAACATTCCATACAATGTGGCATGTCACACGTGGCGCTGTTCTAATGCATAAAGGAAAGAGGATTGAACCATCATGGGCGGACGTCAAGAAAGACCTAATATCATATGGAGGAGGCTGGAAGTTAGAAGGAGAATGGAAGGAAGGAGAAGAAGTCCAGGTATTGGCACTGGAGCCTGGAAAAAATCCAAGAGCCGTCCAAACGAAACCTGGTCTTTTCAAAACCAACGCCGGAACAATAGGTGCTGTATCTCTGGACTTTTCTCCTGGAACGTCAGGATCTCCAATTATCGACAAAAAAGGAAAAGTTGTGGGTCTTTATGGTAATGGTGTTGTTACAAGGAGTGGAGCATATGTGAGTGCTATAGCCCAGACTGAAAAAAGCATTGAAGACAACCCAGAGATCGAAGATGACATTTTCCGAAAGAGAAGACTGACCATCATGGACCTCCACCCAGGAGCGGGAAAGACGAAGAGATACCTTCCGGCCATAGTCAGAGAAGCTATAAAACGGGGTTTGAGAACATTAATCTTGGCCCCCACTAGAGTTGTGGCAGCTGAAATGGAGGAAGCCCTTAGAGGACTTCCAATAAGATACCAGACCCCAGCCATCAGAGCTGAGCACACCGGGCGGGAGATTGTGGACCTAATGTGTCATGCCACATTTACCATGAGGCTGCTATCACCAGTTAGAGTGCCAAACTACAACCTGATTATCATGGACGAAGCCCATTTCACAGACCCAGCAAGTATAGCAGCTAGAGGATACATCTCAACTCGAGTGGAGATGGGTGAGGCAGCTGGGATTTTTATGACAGCCACTCCCCCGGGAAGCAGAGACCCATTTCCTCAGAGCAATGCACCAATCATAGATGAAGAAAGAGAAATCCCTGAACGTTCGTGGAATTCCGGACATGAATGGGTCACGGATTTTAAAGGGAAGACTGTTTGGTTCGTTCCAAGTATAAAAGCAGGAAATGATATAGCAGCTTGCCTGAGGAAAAATGGAAAGAAAGTGATACAACTCAGTAGGAAGACCTTTGATTCTGAGTATGTCAAGACTAGAACCAATGATTGGGACTTCGTGGTTACAACTGACATTTCAGAAATGGGTGCCAATTTCAAGGCTGAGAGGGTTATAGACCCCAGACGCTGCATGAAACCAGTCATACTAACAGATGGTGAAGAGCGGGTGATTCTGGCAGGACCTATGCCAGTGACCCACTCTAGTGCAGCACAAAGAAGAGGGAGAATAGGAAGAAATCCAAAAAATGAGAATGACCAGTACATATACATGGGGGAACCTCTGGAAAATGATGAAGACTGTGCACACTGGAAAGAAGCTAAAATGCTCCTAGATAACATCAACACGCCAGAAGGAATCATTCCTAGCATGTTCGAACCAGAGCGTGAAAAGGTGGATGCCATTGATGGCGAATACCGCTTGAGAGGAGAAGCAAGGAAAACCTTTGTAGACTTAATGAGAAGAGGAGACCTACCAGTCTGGTTGGCCTACAGAGTGGCAGCTGAAGGCATCAACTACGCAGACAGAAGGTGGTGTTTTGATGGAGTCAAGAACAACCAAATCCTAGAAGAAAACGTGGAAGTTGAAATCTGGACAAAAGAAGGGGAAAGGAAGAAATTGAAACCCAGATGGTTGGATGCTAGGATCTATTCTGACCCACTGGCGCTAAAAGAATTTAAGGAATTTGC AGCCGGAAGAAAG19. DEN-2 E protein amino acid sequence with mutations (SEQ ID NO: 19)MRCIGMSNRDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEAKQPATLRKYCIEAKLTNTTTESRCPTQGEPSLNEEQDKRFVCKHSMVDRGWGNGCGLFGKGGIVTCAMFRCKKNMEGKVVQPENLEYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSITEAELTGYGTVTMECSPRTGLDFNEMVLLQMENKAWLVHRQWFLDLPLPWLPGADTQGSNWIQKETLVTFKNPHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFKVVKEIAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLNWFKKGSSLGQAFETTMRGAKRLAILGDTAWDFGSLGGVFTSIGKALHQVFGAIYGAAFSGVSWTMKILIGVIITWIGMNSRSTSLSVTLVLVGIVTLYLGVMVQA20. ZIKV CprME wild-type nucleotide sequence (SEQ ID NO: 20)ATGAAAAACCCAAAAAAGAAATCCGGAGGATTCCGGATTGTCAATATGCTAAAACGCGGAGTAGCCCGTGTGAGCCCCTTTGGGGGCTTGAAGAGGCTGCCAGCCGGACTTCTGCTGGGTCATGGGCCCATCAGGATGGTCTTGGCGATTCTAGCCTTTTTGAGATTCACGGCAATCAAGCCATCACTGGGTCTCATCAATAGATGGGGTTCAGTGGGGAAAAAAGAGGCTATGGAAATAATAAAGAAGTTCAAGAAAGATCTGGCTGCCATGCTGAGAATAATCAATGCTAGGAAGGAGAAGAAGAGACGAGGCGCAGATACTAGTGTCGGAATTGTTGGCCTCCTGCTGACCACAGCTATGGCAGCGGAGGTCACTAGACGTGGGAGTGCATACTATATGTACTTGGACAGAAACGACGCTGGGGAGGCCATATCTTTTCCAACCACATTGGGGATGAATAAGTGTTATATACAGATCATGGATCTTGGACACATGTGTGATGCCACCATGAGCTATGAATGCCCTATGCTGGATGAGGGGGTGGAACCAGATGACGTCGATTGTTGGTGCAACACGACGTCAACTTGGGTTGTGTACGGAACCTGCCATCACAAAAAAGGTGAAGCACGGAGATCTAGAAGAGCTGTGACGCTCCCCTCCCATTCCACTAGGAAGCTGCAAACGCGGTCGCAAACCTGGTTGGAATCAAGAGAATACACAAAGCACTTGATTAGAGTCGAAAATTGGATATTCAGGAACCCTGGCTTCGCGTTAGCAGCAGCTGCCATCGCTTGGCTTTTGGGAAGCTCAACGAGCCAAAAAGTCATATACTTGGTCATGATACTGCTGATTGCCCCGGCATACAGCATCAGGTGCATAGGAGTCAGCAATAGGGACTTTGTGGAAGGTATGTCAGGTGGGACTTGGGTTGATGTTGTCTTGGAACATGGAGGTTGTGTCACCGTAATGGCACAGGACAAACCGACTGTCGACATAGAGCTGGTTACAACAACAGTCAGCAACATGGCGGAGGTAAGATCCTACTGCTATGAGGCATCAATATCGGACATGGCTTCGGACAGCCGCTGCCCAACACAAGGTGAAGCCTACCTTGACAAGCAATCAGACACTCAATATGTCTGCAAAAGAACGTTAGTGGACAGAGGCTGGGGAAATGGATGTGGACTTTTTGGCAAAGGGAGCCTGGTGACATGCGCTAAGTTTGCATGCTCCAAGAAAATGACCGGGAAGAGCATCCAGCCAGAGAATCTGGAGTACCGGATAATGCTGTCAGTTCATGGCTCCCAGCACAGTGGGATGATCGTTAATGACACAGGACATGAAACTGATGAGAATAGAGCGAAGGTTGAGATAACGCCCAATTCACCAAGAGCCGAAGCCACCCTGGGGGGTTTTGGAAGCCTAGGACTTGATTGTGAACCGAGGACAGGCCTTGACTTTTCAGATTTGTATTACTTGACTATGAATAACAAGCACTGGTTGGTTCACAAGGAGTGGTTCCACGACATTCCATTACCTTGGCACGCTGGGGCAGACACCGGAACTCCACACTGGAACAACAAAGAAGCACTGGTAGAGTTCAAGGACGCACATGCCAAAAGGCAAACTGTCGTGGTTCTAGGGAGTCAAGAAGGAGCAGTTCACACGGCCCTTGCTGGAGCTCTGGAGGCTGAGATGGATGGTGCAAAGGGAAGGCTGTCCTCTGGCCACTTGAAATGTCGCCTGAAAATGGATAAACTTAGATTGAAGGGCGTGTCATACTCCTTGTGTACCGCAGCGTTCACATTCACCAAGATCCCGGCTGAAACACTGCACGGGACAGTCACAGTGGAGGTACAGTACGCAGGGACAGATGGACCTTGCAAGGTTCCAGCTCAGATGGCGGTGGACATGCAAACTCTGACCCCAGTTGGGAGGTTGATAACCGCTAACCCCGTAATCACTGAAAGCACTGAGAACTCTAAGATGATGCTGGAACTTGATCCACCATTTGGGGACTCTTACATTGTCATAGGAGTCGGGGAGAAGAAGATCACCCACCACTGGCACAGGAGTGGCAGCACCATTGGAAAAGCATTTGAAGCCACTGTGAGAGGTGCCAAGAGAATGGCAGTCTTGGGAGACACAGCCTGGGACTTTGGATCAGTTGGAGGCGCTCTCAACTCATTGGGCAAGGGCATCCATCAAATTTTTGGAGCAGCTTTCAAATCATTGTTTGGAGGAATGTCCTGGTTCTCACAAATTCTCATTGGAACGTTGCTGATGTGGTTGGGTCTGAACACAAAGAATGGATCTATTTCCCTTATGTGCTTGGCCTTAGGGGGAGTGTTGATCTTCTTATCCACAGCTGTCTCTGCT21. ZIKV CprME codon optimized nucleotide sequence (SEQ ID NO: 21)ATGAAGAACCCCAAGAAGAAGTCCGGCGGCTTCCGGATCGTGAACATGCTGAAGAGAGGCGTGGCCAGAGTCAGCCCCTTCGGCGGACTGAAAAGACTGCCTGCCGGACTGCTGCTGGGCCACGGCCCTATTAGAATGGTGCTGGCCATCCTGGCCTTTCTGCGGTTCACCGCCATCAAGCCCTCCCTGGGCCTGATCAACAGATGGGGCAGCGTGGGCAAGAAAGAAGCCATGGAAATCATCAAGAAGTTCAAGAAAGACCTGGCCGCCATGCTGCGGATCATCAACGCCCGGAAAGAGAAGAAGCGCAGAGGCGCCGATACCTCCGTGGGCATTGTGGGCCTGCTGCTGACAACAGCCATGGCCGCCGAAGTGACCAGAAGAGGCAGCGCCTACTACATGTACCTGGACCGGAATGACGCCGGCGAGGCCATCAGCTTTCCAACCACCCTGGGCATGAACAAGTGCTACATCCAGATCATGGACCTGGGCCACATGTGCGACGCCACAATGAGCTACGAGTGCCCCATGCTGGACGAGGGCGTGGAACCCGACGATGTGGACTGCTGGTGCAACACCACCAGCACCTGGGTGGTGTACGGCACCTGTCACCACAAGAAGGGCGAAGCCCGCAGATCCAGACGGGCCGTGACACTGCCTAGCCACAGCACCAGAAAGCTGCAGACCAGAAGCCAGACCTGGCTGGAAAGCAGAGAGTACACCAAGCACCTGATCCGGGTGGAAAACTGGATCTTCCGGAACCCCGGCTTTGCCCTGGCCGCTGCTGCTATTGCTTGGCTGCTGGGAAGCAGCACCAGCCAGAAAGTGATCTACCTGGTCATGATCCTGCTGATCGCCCCTGCCTACAGCATCCGGTGCATCGGCGTGTCCAACCGGGACTTCGTGGAAGGCATGAGCGGCGGCACATGGGTGGACGTGGTGCTGGAACACGGCGGCTGTGTGACCGTGATGGCCCAGGATAAGCCCACCGTGGACATCGAGCTGGTCACCACCACCGTGTCCAATATGGCCGAAGTGCGGAGCTACTGCTACGAGGCCAGCATCAGCGACATGGCCAGCGACAGCAGATGCCCTACACAGGGCGAGGCCTACCTGGACAAGCAGTCCGACACCCAGTACGTGTGCAAGCGGACCCTGGTGGACAGAGGCTGGGGCAATGGCTGCGGCCTGTTTGGCAAGGGCAGCCTCGTGACCTGCGCCAAGTTCGCCTGCAGCAAGAAGATGACCGGCAAGAGCATCCAGCCCGAGAACCTGGAATACCGGATCATGCTGAGCGTGCACGGCAGCCAGCACTCCGGCATGATCGTGAATGACACCGGCCACGAGACAGACGAGAACCGGGCCAAGGTGGAAATCACCCCTAACAGCCCTAGAGCCGAGGCCACACTGGGCGGCTTTGGATCTCTGGGCCTGGACTGCGAGCCCCGGACCGGCCTGGATTTCAGCGACCTGTACTACCTGACCATGAACAACAAGCACTGGCTGGTCCACAAAGAGTGGTTCCACGACATCCCTCTGCCCTGGCATGCCGGCGCTGATACAGGCACCCCTCACTGGAACAACAAAGAGGCCCTGGTCGAGTTCAAGGACGCCCACGCCAAGAGGCAGACAGTGGTGGTCCTGGGATCTCAGGAAGGCGCCGTCCATACAGCTCTGGCTGGCGCCCTGGAAGCCGAGATGGATGGCGCTAAGGGCAGACTGTCCAGCGGCCACCTGAAGTGCCGGCTGAAGATGGACAAGCTGCGGCTGAAGGGCGTGTCCTACAGCCTGTGTACCGCCGCCTTCACCTTCACCAAGATCCCCGCCGAGACACTGCACGGCACCGTGACCGTGGAAGTGCAGTATGCCGGCACCGATGGCCCATGCAAGGTGCCAGCTCAGATGGCCGTGGATATGCAGACCCTGACCCCTGTGGGCCGGCTGATCACCGCCAATCCTGTGATCACCGAGAGCACCGAGAACAGCAAGATGATGCTGGAACTGGACCCTCCATTCGGCGACAGCTACATCGTGATCGGAGTGGGCGAGAAGAAGATCACCCACCACTGGCACAGAAGCGGCAGCACCATCGGCAAGGCCTTCGAGGCTACAGTGCGGGGAGCCAAGAGAATGGCCGTGCTGGGCGATACCGCCTGGGATTTTGGTTCTGTGGGCGGAGCCCTGAACAGCCTGGGCAAGGGAATCCACCAGATCTTCGGAGCCGCCTTTAAGAGCCTGTTCGGCGGCATGTCCTGGTTCAGCCAGATCCTGATCGGCACCCTGCTGATGTGGCTGGGACTGAACACCAAGAACGGCAGCATCTCCCTGATGTGCCTGGCCCTGGGCGGCGTGCTGATCTTTCTGAGCACAGCCGTGTCCGCCTGA22. ZIKV CprME amino acid sequence (SEQ ID NO: 22)MKNPKKKSGGFRIVNMLKRGVARVSPFGGLKRLPAGLLLGHGPIRMVLAILAFLRFTAIKPSLGLINRWGSVGKKEAMEIIKKFKKDLAAMLRIINARKEKKRRGADTSVGIVGLLLTTAMAAEVTRRGSAYYMYLDRNDAGEAISFPTTLGMNKCYIQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLMWLGLNTKNGSISLMCLALGGVLIFLSTAVSA23. ZIKV NS2B/NS3 Full length nucleotide sequence (SEQ ID NO: 23)AGCTGGCCCCCTAGCGAAGTACTCACAGCTGTTGGCCTGATATGCGCATTGGCTGGAGGGTTCGCCAAGGCAGATATAGAGATGGCTGGGCCCATGGCCGCGGTCGGTCTGCTAATTGTCAGTTACGTGGTCTCAGGAAAGAGTGTGGACATGTACATTGAAAGAGCAGGTGACATCACATGGGAAAAAGATGCGGAAGTCACTGGAAACAGTCCCCGGCTCGATGTGGCGCTAGATGAGAGTGGTGATTTCTCCCTGGTGGAGGATGACGGTCCCCCCATGAGAGAGATCATACTCAAGGTGGTCCTGATGACCATCTGTGGCATGAACCCAATAGCCATACCCTTTGCAGCTGGAGCGTGGTACGTATACGTGAAGACTGGAAAAAGGAGTGGTGCTCTATGGGATGTGCCTGCTCCCAAGGAAGTAAAAAAGGGGGAGACCACAGATGGAGTGTACAGAGTAATGACTCGTAGACTGCTAGGTTCAACACAAGTTGGAGTGGGAGTTATGCAAGAGGGGGTCTTTCACACTATGTGGCACGTCACAAAAGGATCCGCGCTGAGAAGCGGTGAAGGGAGACTTGATCCATACTGGGGAGATGTCAAGCAGGATCTGGTGTCATACTGTGGTCCATGGAAGCTAGATGCCGCCTGGGACGGGCACAGCGAGGTGCAGCTCTTGGCCGTGCCCCCCGGAGAGAGAGCGAGGAACATCCAGACTCTGCCCGGAATATTTAAGACAAAGGATGGGGACATTGGAGCGGTTGCGCTGGATTACCCAGCAGGAACTTCAGGATCTCCAATCCTAGACAAGTGTGGGAGAGTGATAGGACTTTATGGCAATGGGGTCGTGATCAAAAATGGGAGTTATGTTAGTGCCATCACCCAAGGGAGGAGGGAGGAAGAGACTCCTGTTGAGTGCTTCGAGCCTTCGATGCTGAAGAAGAAGCAGCTAACTGTCTTAGACTTGCATCCTGGAGCTGGGAAAACCAGGAGAGTTCTTCCTGAAATAGTCCGTGAAGCCATAAAAACAAGACTCCGTACTGTGATCTTAGCTCCAACCAGGGTTGTCGCTGCTGAAATGGAGGAAGCCCTTAGAGGGCTTCCAGTGCGTTATATGACAACAGCAGTCAATGTCACCCACTCTGGAACAGAAATCGTCGACTTAATGTGCCATGCCACCTTCACTTCACGTCTACTACAGCCAATCAGAGTCCCCAACTATAATCTGTATATTATGGATGAGGCCCACTTCACAGATCCCTCAAGTATAGCAGCAAGAGGATACATTTCAACAAGGGTTGAGATGGGCGAGGCGGCTGCCATCTTCATGACCGCCACGCCACCAGGAACCCGTGACGCATTTCCGGACTCCAACTCACCAATTATGGACACCGAAGTGGAAGTCCCAGAGAGAGCCTGGAGCTCAGGCTTTGATTGGGTGACGGATCATTCTGGAAAAACAGTTTGGTTTGTTCCAAGCGTGAGGAACGGCAATGAGATCGCAGCTTGTCTGACAAAGGCTGGAAAACGGGTCATACAGCTCAGCAGAAAGACTTTTGAGACAGAGTTCCAGAAAACAAAACATCAAGAGTGGGACTTTGTCGTGACAACTGACATTTCAGAGATGGGCGCCAACTTTAAAGCTGACCGTGTCATAGATTCCAGGAGATGCCTAAAGCCGGTCATACTTGATGGCGAGAGAGTCATTCTGGCTGGACCCATGCCTGTCACACATGCCAGCGCTGCCCAGAGGAGGGGGCGCATAGGCAGGAATCCCAACAAACCTGGAGATGAGTATCTGTATGGAGGTGGGTGCGCAGAGACTGACGAAGACCATGCACACTGGCTTGAAGCAAGAATGCTCCTTGACAATATTTACCTCCAAGATGGCCTCATAGCCTCGCTCTATCGACCTGAGGCCGACAAAGTAGCAGCCATTGAGGGAGAGTTCAAGCTTAGGACGGAGCAAAGGAAGACCTTTGTGGAACTCATGAAAAGAGGAGATCTTCCTGTTTGGCTGGCCTATCAGGTTGCATCTGCCGGAATAACCTACACAGATAGAAGATGGTGCTTTGATGGCACGACCAACAACACCATAATGGAAGACAGTGTGCCGGCAGAGGTGTGGACCAGACACGGAGAGAAAAGAGTGCTCAAACCGAGGTGGATGGACGCCAGAGTTTGTTCAGATCATGCGGCCCTGAAGTCATTCAAGGAGTTTGCCGCTGGGAAAAGA24. ZIK NS2B/NS3 full length amino acid sequence (SEQ ID NO: 24)MSWPPSEVLTAVGLICALAGGFAKADIEMAGPMAAVGLLIVSYVVSGKSVDMYIERAGDITWEKDAEVTGNSPRLDVALDESGDFSLVEDDGPPMREIILKVVLMTICGMNPIAIPFAAGAWYVYVKTGKRSGALWDVPAPKEVKKGETTDGVYRVMTRRLLGSTQVGVGVMQEGVFHTMWHVTKGSALRSGEGRLDPYWGDVKQDLVSYCGPWKLDAAWDGHSEVQLLAVPPGERARNIQTLPGIFKTKDGDIGAVALDYPAGTSGSPILDKCGRVIGLYGNGVVIKNGSYVSAITQGRREEETPVECFEPSMLKKKQLTVLDLHPGAGKTRRVLPEIVREAIKTRLRTVILAPTRVVAAEMEEALRGLPVRYMTTAVNVTHSGTEIVDLMCHATFTSRLLQPIRVPNYNLYIMDEAHFTDPSSIAARGYISTRVEMGEAAAIFMTATPPGTRDAFPDSNSPIMDTEVEVPERAWSSGFDWVTDHSGKTVWFVPSVRNGNEIAACLTKAGKRVIQLSRKTFETEFQKTKHQEWDFVVTTDISEMGANFKADRVIDSRRCLKPVILDGERVILAGPMPVTHASAAQRRGRIGRNPNKPGDEYLYGGGCAETDEDHAHWLEARMLLDNIYLQDGLIASLYRPEADKVAAIEGEFKLRTEQRKTFVELMKRGDLPVWLAYQVASAGITYTDRRWCFDGTTNNTIMEDSVPAEVWTRHGEKRVLKPRWMDARVCSDHAALKSFKEFAAGKR25. ZIK NS2B/NS3Pro codon optimized nucleotide sequence (SEQ ID NO: 25)ATGTCTTGGCCTCCATCTGAGGTGCTGACCGCCGTGGGACTGATTTGTGCCCTGGCTGGCGGATTCGCCAAGGCCGACATTGAGATGGCCGGACCTATGGCCGCTGTGGGCCTGCTGATCGTGTCCTACGTGGTGTCCGGCAAGAGCGTGGACATGTACATCGAGAGAGCCGGCGACATCACCTGGGAGAAGGATGCCGAAGTGACCGGCAACAGCCCCAGACTGGATGTGGCCCTGGACGAGAGCGGCGATTTCAGCCTGGTGGAAGATGACGGCCCTCCCATGCGCGAGATCATCCTGAAGGTGGTGCTGATGACCATCTGCGGAATGAACCCTATCGCCATCCCCTTCGCCGCTGGCGCTTGGTACGTGTACGTGAAAACCGGCAAGCGGAGCGGAGCCCTGTGGGACGTGCCAGCCCCTAAAGAAGTGAAGAAGGGCGAGACAACCGACGGCGTGTACAGAGTGATGACCAGACGGCTGCTGGGCAGCACACAAGTCGGAGTGGGAGTGATGCAGGAAGGGGTCTTCCACACCATGTGGCACGTGACCAAGGGCAGCGCCCTGAGATCTGGCGAAGGCAGACTGGACCCTTACTGGGGCGACGTGAAGCAGGACCTGGTGTCCTACTGCGGCCCCTGGAAACTGGATGCCGCCTGGGATGGCCACAGCGAAGTGCAGCTGCTGGCTGTGCCTCCCGGCGAGAGGGCCAGAAATATCCAGACCCTGCCCGGCATCTTCAAGACCAAGGATGGCGACATCGGCGCCGTGGCTCTGGATTACCCTGCCGGCACATCTGGCAGCCCCATCCTGGATAAGTGCGGCAGAGTGATCGGCCTGTACGGCAACGGCGTGGTCATCAAGAACGGCAGCTACGTGTCCGCCATCACCCAGGGCAGACGCGAGGAAGAGACACCCGTGGAATGCTTCGAGTGA26. ZIKV NS2B/NS3Pro amino acid sequence (SEQ ID NO: 26)MSWPPSEVLTAVGLICALAGGF AKADIEMAGPMAAVGLLIVSYVVSGKSVDMYIERAGDITWEKDAEVTGNSPRLDVALDESGDFSLVEDDGPPMREIILKVVLMTICGMNPIAIPFAAGAWYVYVKTGKRSGALWDVPAPKEVKKGETTDGVYRVMTRRLLGSTQVGVGVMQEGVFHTMWHVTKGSALRSGEGRLDPYWGDVKQDLVSYCGPWKLDAAWDGHSEVQLLAVPPGERARNIQTLPGIFKTKDGDIGAVALDYPAGTSGSPILDKCGRVIGLYGNGVVIKNGSYVSAITQGRREEET PVECFE27. DEN-2 NS3 wild-type amino acid sequence (SEQ ID NO: 27)AGVLWDVPSPPPMGKAELEDGAYRIKQKGILGYSQIGAGVYKEGTFHTMWHVTRGAVLMHKGKRIEPSWADVKKDLISYGGGWKLEGEWKEGEEVQVLALEPGKNPRAVQTKPGLFKTNAGTIGAVSLDFSPGTSGSPIIDKKGKVVGLYGNGVVTRSGAYVSAIAQTEKSIEDNPEIEDDIFRKRRLTIMDLHPGAGKTKRYLPAIVREAIKRGLRTLILAPTRVVAAEMEEALRGLPIRYQTPAIRAEHTGREIVDLMCHATFTMRLLSPVRVPNYNLIIMDEAHFTDPASIAARGYISTRVEMGEAAGIFMTATPPGSRDPFPQSNAPIIDEEREIPERSWNSGHEWVTDFKGKTVWFVPSIKAGNDIAACLRKNGKKVIQLSRKTFDSEYVKTRTNDWDFVVTTDISEMGANFKAERVIDPRRCMKPVILTDGEERVILAGPMPVTHSSAAQRRGRIGRNPKNENDQYIYMGEPLENDEDCAHWKEAKMLLDNINTPEGIIPSMFEPEREKVDAIDGEYRLRGEARKTFVDLMRRGDLPVWLAYRVAAEGINYADRRWCFDGVKNNQILEENVEVEIWTKEGERKKLKPRWLDARIYSDPLALKEFKE FAAGR28. DEN-2 NS3 Helicase portion wild type nucleotide sequence(SEQ ID NO: 28) ATTTTCCGAAAGAGAAGACTGACCATCATGGACCTCCACCCAGGAGCGGGAAAGACGAAGAGATACCTTCCGGCCATAGTCAGAGAAGCTATAAAACGGGGTTTGAGAACATTAATCTTGGCCCCCACTAGAGTTGTGGCAGCTGAAATGGAGGAAGCCCTTAGAGGACTTCCAATAAGATACCAGACCCCAGCCATCAGAGCTGAGCACACCGGGCGGGAGATTGTGGACCTAATGTGTCATGCCACATTTACCATGAGGCTGCTATCACCAGTTAGAGTGCCAAACTACAACCTGATTATCATGGACGAAGCCCATTTCACAGACCCAGCAAGTATAGCAGCTAGAGGATACATCTCAACTCGAGTGGAGATGGGTGAGGCAGCTGGGATTTTTATGACAGCCACTCCCCCGGGAAGCAGAGACCCATTTCCTCAGAGCAATGCACCAATCATAGATGAAGAAAGAGAAATCCCTGAACGTTCGTGGAATTCCGGACATGAATGGGTCACGGATTTTAAAGGGAAGACTGTTTGGTTCGTTCCAAGTATAAAAGCAGGAAATGATATAGCAGCTTGCCTGAGGAAAAATGGAAAGAAAGTGATACAACTCAGTAGGAAGACCTTTGATTCTGAGTATGTCAAGACTAGAACCAATGATTGGGACTTCGTGGTTACAACTGACATTTCAGAAATGGGTGCCAATTTCAAGGCTGAGAGGGTTATAGACCCCAGACGCTGCATGAAACCAGTCATACTAACAGATGGTGAAGAGCGGGTGATTCTGGCAGGACCTATGCCAGTGACCCACTCTAGTGCAGCACAAAGAAGAGGGAGAATAGGAAGAAATCCAAAAAATGAGAATGACCAGTACATATACATGGGGGAACCTCTGGAAAATGATGAAGACTGTGCACACTGGAAAGAAGCTAAAATGCTCCTAGATAACATCAACACGCCAGAAGGAATCATTCCTAGCATGTTCGAACCAGAGCGTGAAAAGGTGGATGCCATTGATGGCGAATACCGCTTGAGAGGAGAAGCAAGGAAAACCTTTGTAGACTTAATGAGAAGAGGAGACCTACCAGTCTGGTTGGCCTACAGAGTGGCAGCTGAAGGCATCAACTACGCAGACAGAAGGTGGTGTTTTGATGGAGTCAAGAACAACCAAATCCTAGAAGAAAACGTGGAAGTTGAAATCTGGACAAAAGAAGGGGAAAGGAAGAAATTGAAACCCAGATGGTTGGATGCTAGGATCTATTCTGACCCACTGGCGCTAAAAGAATTTAAGGAATT TGCAGCCGGAAGAAAG29. DEN-2 NS3 Helicase wild type amino acid sequence (SEQ ID NO: 29)IFRKRRLTIMDLHPGAGKTKRYLPAIVREAIKRGLRTLILAPTRVVAAEMEEALRGLPIRYQTPAIRAEHTGREIVDLMCHATFTMRLLSPVRVPNYNLIIMDEAHFTDPASIAARGYISTRVEMGEAAGIFMTATPPGSRDPFPQSNAPIIDEEREIPERSWNSGHEWVTDFKGKTVWFVPSIKAGNDIAACLRKNGKKVIQLSRKTFDSEYVKTRTNDWDFVVTTDISEMGANFKAERVIDPRRCMKPVILTDGEERVILAGPMPVTHSSAAQRRGRIGRNPKNENDQYIYMGEPLENDEDCAHWKEAKMLLDNINTPEGIIPSMFEPEREKVDAIDGEYRLRGEARKTFVDLMRRGDLPVWLAYRVAAEGINYADRRWCFDGVKNNQILEENVEVEIWTKEGERKKLKPRWLDARIYSDPLALKEFKEFAAGR30. DEN-2 NS2B/NS3Pro wild-type nucleotide sequence with Mutations(SEQ ID NO: 30) ATGAGCTGGCCATTAAATGAGGCTATCATGGCAGTCGGGATGGTGAGCATTTTAGCCAGTTCTCTCCTAAAAAATGATATTCCCATGACAGGACCATTAGTGGCTGGAGGGCTCCTCACTGTGTGCTACGTGCTCACTGGACGATCGGCCGATTTGGAACTGGAGAGAGCAGCCGATGTCAAATGGGAAGACCAGGCAGAGATATCAGGAAGCAGTCCAATCCTGTCAATAACAATATCAGAAGATGGTAGCATGTCGATAAAAAATGAAGAGGAAGAACAAACACTGACCATACTCATTAGAACAGGATTGCTGGTGATCTCAGGACTTTTTCCTGTATCAATACCAATCACGGCAGCAGCATGGTACCTGTGGGAAGTGAAGAAACAACGGGCCGGAGTATTGTGGGATGTTCCTTCACCCCCACCCATGGGAAAGGCTGAACTGGAAGATGGAGCCTATAGAATTAAGCAAAAAGGGATTCTTGGATATTCCCAGATCGGAGCCGGAGTTTACAAAGAAGGAACATTCCATACAATGTGGCATGTCACACGTGGCGCTGTTCTAATGCATAAAGGAAAGAGGATTGAACCATCATGGGCGGACGTCAAGAAAGACCTAATATCATATGGAGGAGGCTGGAAGTTAGAAGGAGAATGGAAGGAAGGAGAAGAAGTCCAGGTATTGGCACTGGAGCCTGGAAAAAATCCAAGAGCCGTCCAAACGAAACCTGGTGCCTTCAAAACCAACGCCGGAACAATAGGTGCTGTATCTCTGGACTTTTCTCCTGGAACGTCAGGATCTCCAATTATCGACAAAAAAGGAAAAGTTGTGGGTCTTTATGGTAATGGTGTTGTTACAAGGAGTGGAGCATATGTGAGTGCTATAGCCCAGACTGAAAAAAGCATTGAAGACAACCCAGAGATCGAAGATGACTAG31. DEN-2 Capsid (C) codon optimized nucleotide sequence (SEQ ID NO: 31)ATGAACAACCAGCGCAAGAAGGCCAAAAACACTCCGTTCAATATGCTCAAGAGAGAGCGCAATCGGGTTTCTACGGTACAGCAGCTGACGAAGAGATTCTCCCTGGGCATGCTGCAAGGTCGCGGACCACTGAAGCTGTTCATGGCCCTTGTTGCATTTCTTAGGTTTCTTACAATTCCCCCCACTGCTGGAATCCTGAAGCGGTGGGGCACCATCAAAAAGTCCAAGGCTATTAATGTCCTCAGGGGGTTCAGGAAAGAGATTGGGCGGATGCTGAACATCCTTAATAGACGCAGACGGTCCGCTGGCATGATAATCATGCTGATCCCAACCGTCATGGCC32. DEN-2 Capsid (C) amino acid sequence (SEQ ID NO: 32)MNNQRKKAKNTPFNMLKRERNRVSTVQQLTKRFSLGMLQGRGPLKLFMALVAFLRFLTIPPTAGILKRWGTIKKSKAINVLRGFRKEIGRMLNILNRRRRSA GMIIMLIPTVMADengue virus 1 Strain West Pac 94 Genbank M23027.133. DEN-1 prME wild type nucleotide sequence (SEQ ID NO: 33)TTCCATCTGACCACCCGAGGGGGAGAGCCGCACATGATAGTTAGCAAGCAGGAAAGAGGAAAATCACTTTTGTTTAAGACCTCTGCAGGTGTCAACATGTGCACCCTTATTGCAATGGATTTGGGAGAGTTATGTGAGGACACAATGACCTACAAATGCCCCCGGATCACTGAGACGGAACCAGATGACGTTGACTGTTGGTGCAATGCCACGGAGACATGGGTGACCTATGGAACATGTTCTCAAACTGGTGAACACCGACGAGACAAACGTTCCGTCGCACTGGCACCACACGTAGGGCTTGGTCTAGAAACAAGAACCGAAACGTGGATGTCCTCTGAAGGCGCTTGGAAACAAATACAAAAAGTGGAGACCTGGGCTCTGAGACACCCAGGATTCACGGTGATAGCCCTTTTTCTAGCACATGCCATAGGAACATCCATCACCCAGAAAGGGATCATTTTTATTTTGCTGATGCTGGTAACTCCATCCATGGCCATGCGGTGCGTGGGAATAGGCAACAGAGACTTCGTGGAAGGACTGTCAGGAGCTACGTGGGTGGATGTGGTACTGGAGCATGGAAGTTGCGTCACTACCATGGCAAAAGACAAACCAACACTGGACATTGAACTCTTGAAGACGGAGGTCACAAACCCTGCCGTCCTGCGCAAACTGTGCATTGAAGCTAAAATATCAAACACCACCACCGATTCGAGATGTCCAACACAAGGAGAAGCCACGCTGGTGGAAGAACAGGACACGAACTTTGTGTGTCGACGAACGTTCGTGGACAGAGGCTGGGGCAATGGTTGTGGGCTATTCGGAAAAGGTAGCTTAATAACGTGTGCTAAGTTTAAGTGTGTGACAAAACTGGAAGGAAAGATAGTCCAATATGAAAACTTAAAATATTCAGTGATAGTCACCGTACACACTGGAGACCAGCACCAAGTTGGAAATGAGACCACAGAACATGGAACAACTGCAACCATAACACCTCAAGCTCCCACGTCGGAAATACAGCTGACAGACTACGGAGCTCTAACATTGGATTGTTCACCTAGAACAGGGCTAGACTTTAATGAGATGGTGTTGTTGACAATGGAAAAAAAATCATGGCTCGTCCACAAACAATGGTTTCTAGACTTACCACTGCCTTGGACCTCGGGGGCTTCAACATCCCAAGAGACTTGGAATAGACAAGACTTGCTGGTCACATTTAAGACAGCTCATGCAAAAAAGCAGGAAGTAGTCGTACTAGGATCACAAGAAGGAGCAATGCACACTGCGTTGACTGGAGCGACAGAAATCCAAACGTCTGGAACGACAACAATTTTTGCAGGACACCTGAAATGCAGACTAAAAATGGATAAACTGACTTTAAAAGGGATGTCATATGTAATGTGCACAGGGTCATTCAAGTTAGAGAAGGAAGTGGCTGAGACCCAGCATGGAACTGTTCTAGTGCAGGTTAAATACGAAGGAACAGATGCACCATGCAAGATCCCCTTCTCGTCCCAAGATGAGAAGGGAGTAACCCAGAATGGGAGATTGATAACAGCCAACCCCATAGTCACTGACAAAGAAAAACCAGTCAACATTGAAGCGGAGCCACCTTTTGGTGAGAGCTACATTGTGGTAGGAGCAGGTGAAAAAGCTTTGAAACTAAGCTGGTTCAAGAAGGGAAGCAGTATAGGGAAAATGTTTGAAGCAACTGCCCGTGGAGCACGAAGGATGGCCATCCTGGGAGACACTGCATGGGACTTCGGTTCTATAGGAGGGGTGTTCACGTCTGTGGGAAAACTGATACACCAGATTTTTGGGACTGCGTATGGAGTTTTGTTCAGCGGTGTTTCTTGGACCATGAAGATAGGAATAGGGATTCTGCTGACATGGCTAGGATTAAACTCAAGGAGCACGTCCCTTTCAATGACGTGTATCGCAGTTGGCATGGTCACGCTGTACCTAGGAGTCATGGTTCAGGCG34. DEN-1 prME wild type amino acid sequence (SEQ ID NO: 34)MIVSKQERGKSLLFKTSAGVNMCTLIAMDLGELCEDTMTYKCPRITETEPDDVDCWCNATETWVTYGTCSQTGEHRRDKRSVALAPHVGLGLETRTETWMSSEGAWKQIQKVETWALRHPGFTVIALFLAHAIGTSITQKGIIFILLMLVTPSMAMRCVGIGNRDFVEGLSGATWVDVVLEHGSCVTTMAKDKPTLDIELLKTEVTNPAVLRKLCIEAKISNTTTDSRCPTQGEATLVEEQDTNFVCRRTFVDRGWGNGCGLFGKGSLITCAKFKCVTKLEGKIVQYENLKYSVIVTVHTGDQHQVGNETTEHGTTATITPQAPTSEIQLTDYGALTLDCSPRTGLDFNEMVLLTMEKKSWLVHKQWFLDLPLPWTSGASTSQETWNRQDLLVTFKTAHAKKQEVVVLGSQEGAMHTALTGATEIQTSGTTTIFAGHLKCRLKMDKLTLKGMSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSSQDEKGVTQNGRLITANPIVTDKEKPVNIEAEPPFGESYIVVGAGEKALKLSWFKKGSSIGKMFEATARGARRMAILGDTAWDFGSIGGVFTSVGKLIHQIFGTAYGVLFSGVSWTMKIGIGILLTWLGLNSRSTSLSMTCIAVGMVTLYLGVMVQA35. TVXDO21 (Capsid of DENV-2 and prME of DENV-1)Codon Optimized nucleotide sequence with mutations (SEQ ID NO: 35)ATGAACAACCAGCGCAAGAAGGCCAAAAACACTCCGTTCAATATGCTCAAGAGAGAGCGCAATCGGGTTTCTACGGTACAGCAGCTGACGAAGAGATTCTCCCTGGGCATGCTGCAAGGTCGCGGACCACTGAAGCTGTTCATGGCCCTTGTTGCATTTCTTAGGTTTCTTACAATTCCCCCCACTGCTGGAATCCTGAAGCGGTGGGGCACCATCAAAAAGTCCAAGGCTATTAATGTCCTCAGGGGGTTCAGGAAAGAGATTGGGCGGATGCTGAACATCCTTAATAGACGCAGACGGTCCGCTGGCATGATAATCATGCTGATCCCAACCGTCATGGCCTTTCACCTGACCACTAGGGGCGGTGAGCCACATATGATAGTTAGTAAACAGGAAAGGGGTAAAAGTCTGCTTTTTAAAACTTCCGCCGGCGTAAATATGTGCACACTGATAGCCATGGACTTGGGCGAGCTTTGCGAGGATACCATGACATACAAATGCCCCCGGATCACAGAGACAGAACCAGACGATGTTGACTGCTGGTGCAACGCCACCGAGACTTGGGTTACATACGGGACTTGCAGCCAAACGGGAGAACATAGACGCGCAAAGAGATCTGTAGCCCTTGCCCCACACGTAGGACTGGGACTCGAGACAAGAACAGAAACCTGGATGAGTAGTGAAGGCGCTTGGAAAAGATCCAAAAGGTGGAAACTTGGGCTCTGCGACACCCTGGGTTCACAGTGATCGCATTGTTTTTGGCCCATGCAATAGGAACTTCTATCACACAGAAAGGCATTATCTTCATCCTGCTGATGTTGGTTACACCTTCAATGGCCATGAGGTGCGTCGGTATCGGAAACAGAGATTTCGTGGAAGGGCTGAGCGGGGCTACCTGGGTGGATGTCGTCCTCGAACACGGATCATGTGTCACGACTATGGCAAAAGATAAGCCTACCCTCGATATTGAGCTGTTGAAGACCGAGGTTACTAACCCTGCTGTGCTGCGCAAACTGTGTATTGAAGCAAAGATTTCTAACACAACAACCGACAGTAGATGCCCCACTCAGGGAGAAGCCACGCTGGTGGAAGAGCAGGACACCAACTTTGTATGTAGAAGAACCTTCGTCGATCGCGGATGGGGGAACGGGTGCGGACTCTTCGGAAAAGGATCCCTGATTACTTGTGCAAAATTCAAATGCGTGACTAAACTTGAAGGCAAAATCGTACAGTACGAAAATTTGAAGTACTCTGTTATCGTTACCGTTCATACGGGAGATCAACACCAGGTTGGGAACGAGACCACCGAACACGGCACTACCGCAACGATTACACCTCAAGCCCCTACTTCCGAAATACAACTCACCGACTATGGCGCCCTTACACTGGACTGTTCACCACGCACTGGACTGGACTTCAACGAAATGGTCCTCCTGACAATGGAAAAGAAAAGCTGGCTTGTACACAAGCAATGGTTCTTGGACCTGCCGCTCCCATGGACGAGTGGCGCGAGTACTAGCCAGGAGACCTGGAACCGGCAGGACCTTCTGGTAACATTCAAGACAGCACACGCTAAAAAACAAGAGGTGGTCGTTCTTGGATCCCAAGAGGGTGCAATGCACACAGCCCTCACAGGTGCAACCGAGATCCAGACTTCCGGAACTACCACTATCTTTGCAGGCCATCTCAAATGCAGACTGAAAATGGATAAACTTACACTCAAGGGGATGTCATATGTCATGTGTACGGGGTCTTTTAAACTTGAAAAGGAGGTCGCTGAAACACAACACGGAACTGTTCTGGTGCAAGTCAAATACGAAGGTACGGATGCTCCCTGTAAAATTCCCTTCAGCTCTCAGGACGAAAAAGGTGTTACTCAGAATGGTAGGCTGATTACCGCTAATCCAATTGTAACCGATAAGGAGAAACCCGTGAATATTGAGGCAGAGCCCCCCTTCGGTGAATCTTATATTGTAGTTGGAGCAGGAGAGAAGGCCCTTAAACTCAGTTGGTTCAAGAAGGGATCTTCCCTCGGAAAAGCATTTGAAGCTACGGCTCGGGGAGCGCGCAGGCTGGCTATCCTTGGGGACACGGCATGGGACTTTGGAAGCATTGGTGGCGTCTTTACATCCGTGGGAAAGTTGATACACCAAATCTTCGGGACCGCGTACGGCGTGCTCTTTTCAGGAGTCTCTTGGACTATGAAGATCGGAATTGGCATACTCTTGACCTGGCTTGGCTTGAATTCCCGGTCTACTTCTTTGAGTATGACTTGCATTGCTGTTGGCATGGTCACTCTCTACCTCGGCGTGATGGTGCAGGCCTAG 36. TVXDO21 amino acid sequence with mutations(SEQ ID NO: 36) MNNQRKKAKNTPFNMLKRERNRVSTVQQLTKRFSLGMLQGRGPLKLFMALVAFLRFLTIPPTAGILKRWGTIKKSKAINVLRGFRKEIGRMLNILNRRRRSAGMIIMLIPTVMAFHLTTRGGEPHMIVSKQERGKSLLFKTSAGVNMCTLIAMDLGELCEDTMTYKCPRITETEPDDVDCWCNATETWVTYGTCSQTGEHRRDKRSVALAPHVGLGLETRTETWMSSEGAWKQIQKVETWALRHPGFTVIALFLAHAIGTSITQKGIIFILLMLVTPSMAMRCVGIGNRDFVEGLSGATWVDVVLEHGSCVTTMAKDKPTLDIELLKTEVTNPAVLRKLCIEAKISNTTTDSRCPTQGEATLVEEQDTNFVCRRTFVDRGWGNGCGLFGKGSLITCAKFKCVTKLEGKIVQYENLKYSVIVTVHTGDQHQVGNETTEHGTTATITPQAPTSEIQLTDYGALTLDCSPRTGLDFNEMVLLTMEKKSWLVHKQWFLDLPLPWTSGASTSQETWNRQDLLVTFKTAHAKKQEVVVLGSQEGAMHTALTGATEIQTSGTTTIFAGHLKCRLKMDKLTLKGMSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSSQDEKGVTQNGRLITANPIVTDKEKPVNIEAEPPFGESYIVVGAGEKALKLSWFKKGSSLGKAFEATARGARRLAILGDTAWDFGSIGGVFTSVGKLIHQIFGTAYGVLFSGVSWTMKIGIGILLTWLGLNSRSTSLSMTCIAVGMV TLYLGVMVQADengue virus 3 Strain CH53489 Genbank DQ863638.137. DEN-3 prME wild-type nucleotide sequence (SEQ ID NO: 37)TTCCACTTAACTTCACGAGATGGAGAGCCGCGCATGATTGTGGGGAAGAATGAAAGAGGAAAATCCCTACTTTTTAAGACAGCTTCTGGAATCAACATGTGCACACTCATAGCCATGGACTTGGGAGAGATGTGTGATGACACGGTCACTTACAAATGCCCCCACATTGCCGAAGTGGAACCTGAAGACATTGACTGCTGGTGCAACCTTACATCGACATGGGTGACTTATGGAACGTGCAATCAAGCTGGGGAGCACAGACGCGACAAGAGATCAGTGGCGTTAGCTCCCCATGTCGGCATGGGACTGGACACACGCACCCAAACCTGGATGTCGGCTGAAGGAGCTTGGAGACAAGTCGAGAAGGTAGAGACATGGGCCCTTAGGCACCCAGGGTTCACCATACTAGCTCTATTTCTTGCCCATTACATAGGCACTTCCTTGACCCAGAAAGTGGTTATTTTTATACTACTAATACTGGTCACTCCATCCATGGCAATGAGATGCGTGGGAGTAGGAAACAGAGATTTTGTGGAAGGTCTATCGGGAGCTACGTGGGTTGACGTGGTGCTCGAGCACGGTGGGTGTGTGACCACCATGGCTAAGAACAAGCCCACGCTGGACATAGAGCTTCAGAAGACCGAGGCCACCCAACTGGCCACCCTAAGGAAGTTATGCATTGAGGGAAAAATTACCAACATAACAACTGACTCAAGGTGTCCTACCCAGGGGGAAGCGATTTTACCTGAGGAGCAGGACCAGAACTACGTATGTAAGCATACATACGTGGATAGAGGCTGGGGAAACGGTTGTGGTTTGTTTGGAAAAGGAAGCTTGGTGACATGCGCGAAATTTCAATGCTTAGAATCAATAGAGGGAAAAGTGGTGCAACATGAGAACCTCAAATACACTGTCATCATTACAGTGCACACAGGAGACCAACACCAGGTGGGAAATGAAACGCAGGGAGTCACGGCTGAGATAACACCCCAGGCATCAACCGTTGAAGCTATCTTGCCTGAATATGGAACCCTTGGGCTAGAATGCTCACCACGGACAGGTTTGGATTTCAATGAAATGATCTTATTGACAATGAAGAACAAAGCATGGATGGTACATAGACAATGGTTCTTTGACCTCCCCCTACCATGGACATCAGGAGCTACAACAGAGACACCAACTTGGAACAGGAAAGAGCTTCTTGTGACATTCAAAAATGCACATGCAAAAAAGCAAGAAGTAGTTGTCCTTGGATCGCAAGAGGGAGCAATGCACACAGCGCTGACAGGAGCTACAGAGATCCAAAACTCAGGAGGCACAAGCATTTTTGCCGGGCACTTGAAATGTAGACTTAAGATGGACAAATTGGAACTCAAGGGGATGAGCTATGCAATGTGCTTGAACACCTTTGTGTTGAAGAAAGAAGTCTCCGAGACGCAGCATGGGACAATACTCATTAAGGTTGAGTACAAAGGGGAAGATGCACCTTGCAAGATTCCTTTCTCCACGGAGGATGGACAAGGGAAAGCTCACAATGGTAGACTGATCACAGCCAACCCAGTGGTGACCAAGAAGGAGGAGCCTGTCAACATTGAGGCTGAACCTCCTTTTGGGGAAAGTAACATAGTGATTGGAATTGGAGACAAAGCCTTGAAAATTAACTGGTACAAGAAGGGAAGCTCGATTGGGAAGATGTTCGAGGCCACTGCCAGAGGTGCAAGGCGCATGGCCATCTTGGGAGACACAGCCTGGGACTTTGGATCAGTGGGTGGTGTCTTGAATTCATTAGGGAAAATGGTCCACCAAATATTTGGAAGTGCTTACACAGCCCTGTTTAGTGGAGTCTCATGGATAATGAAAATTGGAATAGGTGTCCTCTTAACCTGGATAGGGTTGAATTCAAAAAACACTTCCATGTCATTTTCATGTATTGCGATAGGAATTATTACACTCTATCTGGGAGCCGTGGTACAAGCT DEN-3 prME wild type amino acid sequence(SEQ ID NO: 48) MIVGKNERGKSLLFKTASGINMCTLIAMDLGEMCDDTVTYKCPHIAEVEPEDIDCWCNLTSTWVTYGTCNQAGEHRRDKRSVALAPHVGMGLDTRTQTWMSAEGAWRQVEKVETWALRHPGFTILALFLAHYIGTSLTQKVVIFILLILVTPSMAMRCVGVGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLDIELQKTEATQLATLRKLCIEGKITNITTDSRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTCAKFQCLESIEGKVVQHENLKYTVIITVHTGDQHQVGNETQGVTAEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWTSGATTETPTWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGATEIQNSGGTSIFAGHLKCRLKMDKLELKGMSYAMCLNTFVLKKEVSETQHGTILIKVEYKGEDAPCKIPFSTEDGQGKAHNGRLITANPVVTKKEEPVNIEAEPPFGESNIVIGIGDKALKINWYKKGSSIGKMFEATARGARRMAILGDTAWDFGSVGGVLNSLGKMVHQIFGSAYTALFSGVSWIMKIGIGVLLTWIGLNSKNTSMSFSCIAIGIITLYLGAVVQA38. TVXDO23 (Capsid of DENV-2 and prME of DENV-3)Codon Optimized nucleotide sequence with mutations (SEQ ID NO: 38)ATGAACAACCAGCGCAAGAAGGCCAAAAACACTCCGTTCAATATGCTCAAGAGAGAGCGCAATCGGGTTTCTACGGTACAGCAGCTGACGAAGAGATTCTCCCTGGGCATGCTGCAAGGTCGCGGACCACTGAAGCTGTTCATGGCCCTTGTTGCATTTCTTAGGTTTCTTACAATTCCCCCCACTGCTGGAATCCTGAAGCGGTGGGGCACCATCAAAAAGTCCAAGGCTATTAATGTCCTCAGGGGGTTCAGGAAAGAGATTGGGCGGATGCTGAACATCCTTAATAGACGCAGACGGTCCGCTGGCATGATAATCATGCTGATCCCAACCGTCATGGCCTTTCATCTGACTTCTCGAGATGGAGAGCCTCGCATGATCGTTGGCAAGAATGAGCGCGGCAAAAGTCTCCTGTTCAAAACGGCCTCTGGAATTAATATGTGTACCTTGATTGCTATGGATCTGGGAGAGATGTGTGATGATACCGTTACCTACAAGTGCCCGCACATTGCTGAGGTTGAGCCTGAAGACATAGACTGCTGGTGCAACTTGACAAGTACGTGGGTCACCTACGGGACCTGCAACCAAGCCGGCGAGCACAGGCGCGCAAAGAGATCCGTTGCGCTGGCGCCACACGTAGGAATGGGCCTGGACACTCGCACTCAGACTTGGATGTCTGCTGAGGGCGCCTGGCGGCAGGTAGAGAAAGTAGAGACATGGGCTCTCAGGCACCCAGGATTTACCATTCTGGCTCTGTTTTTGGCCCACTATATCGGCACCTCCCTCACTCAGAAGGTCGTCATTTTCATACTCCTGATACTCGTGACCCCTTCTATGGCCATGCGGTGTGTCGGGGTCGGCAATAGGGACTTCGTGGAAGGATTGAGTGGCGCAACTTGGGTCGATGTCGTGCTGGAACATGGAGGTTGTGTAACTACTATGGCGAAGAATAAACCAACTCTGGACATCGAGCTGCAAAAGACTGAGGCAACACAACTTGCAACTCTTAGAAAGCTGTGTATCGAAGGCAAAATAACTAATATCACCACAGATTCCAGATGTCCCACCCAGGGGGAAGCTATCCTGCCAGAGGAGCAGGACCAGAATTACGTGTGTAAGCATACCTATGTGGATCGGGGCTGGGGGAATGGATGTGGCCTCTTCGGTAAGGGTTCCCTCGTGACGTGCGCGAAATTCCAGTGTTTGGAATCCATAGAAGGCAAAGTAGTACAACACGAAAACCTCAAATATACAGTTATTATCACTGTTCACACCGGGGACCAGCACCAAGTAGGGAATGAGACACAGGGCGTTACAGCCGAAATTACTCCACAAGCCAGTACAGTCGAGGCTATTCTGCCTGAATATGGTACTTTGGGACTCGAATGCTCACCGCGGACCGGACTGGACTTTAACGAAATGATACTGCTGACAATGAAGAACAAGGCCTGGATGGTACACCGCCAATGGTTCTTTGACCTGCCACTGCCATGGACATCCGGTGCAACAACTGAAACTCCTACATGGAACCGAAAAGAACTGCTCGTCACTTTTAAGAATGCCCATGCTAAAAAACAGGAGGTTGTCGTATTGGGTTCTCAGGAAGGCGCAATGCATACTGCTCTTACAGGGGCCACCGAGATACAAAATTCAGGGGGAACCAGCATCTTCGCAGGGCACTTGAAGTGTAGGCTGAAAATGGACAAGCTGGAGCTCAAGGGAATGAGTTACGCCATGTGCCTCAACACGTTTGTTCTGAAAAAGGAGGTCAGCGAGACACAGCACGGAACAATACTGATTAAGGTTGAGTATAAAGGAGAAGATGCCCCCTGCAAAATTCCTTTCAGCACCGAAGACGGGCAAGGGAAAGCACACAACGGACGCCTGATTACTGCCAATCCCGTCGTCACTAAGAAGGAGGAACCAGTGAATATTGAGGCCGAACCACCTTTTGGGGAATCTAACATTGTAATTGGGATTGGAGACAAAGCATTGAAGATAAATTGGTACAAGAAGGGTTCATCTCTGGGCAAGGCTTTCGAGGCCACAGCGAGAGGGGCAAGACGACTGGCCATTTTGGGGGATACAGCTTGGGACTTCGGTAGCGTCGGCGGAGTGCTGAACTCCCTGGGGAAAATGGTGCACCAGATATTCGGTTCCGCCTACACTGCGCTGTTCTCTGGGGTTAGTTGGATTATGAAAATCGGTATCGGAGTGCTGCTCACGTGGATCGGACTCAACAGTAAGAACACCTCTATGTCATTTAGTTGTATCGCAATTGGAATCATTACCTTGTATCTGGGA GCCGTCGTGCAAGCCTAG39. TVXDO23 amino acid sequence with mutations (SEQ ID NO: 39)FHLTSRDGEPRMIVGKNERGKSLLFKTASGINMCTLIAMDLGEMCDDTVTYKCPHIAEVEPEDIDCWCNLTSTWVTYGTCNQAGEHRRAKRSVALAPHVGMGLDTRTQTWMSAEGAWRQVEKVETWALRHPGFTILALFLAHYIGTSLTQKVVIFILLILVTPSMAMRCVGVGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLDIELQKTEATQLATLRKLCIEGKITNITTDSRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTCAKFQCLESIEGKVVQHENLKYTVIITVHTGDQHQVGNETQGVTAEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWTSGATTETPTWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGATEIQNSGGTSIFAGHLKCRLKMDKLELKGMSYAMCLNTFVLKKEVSETQHGTILIKVEYKGEDAPCKIPFSTEDGQGKAHNGRLITANPVVTKKEEPVNIEAEPPFGESNIVIGIGDKALKINWYKKGSSLGKAFEATARGARRLAILGDTAWDFGSVGGVLNSLGKMVHQIFGSAYTALFSGVSWIMKIGIGVLLTWIGLNSKNTSMSFSCIAIGIITLYLGAVVQADengue virus 4 Strain P75215 Genbank EF45790640. DEN-4 prME wild type nucleotide sequence (SEQ ID NO: 40)TTCCACTTATCGTCAAGAGACGGCGAACCCCTCATGATAGTAGCGAAACACGAAAGGGGGAGACCTCTTTTGTTTAAGACAACGGAAGGAATCAACAGATGCACTCTCATTGCCATGGACGTGGGTGAAATGTGTGAGGACACCGTCACATATAAATGCCCCCTACTGGTCAACACTGAGCCTGAAGACATTGATTGCTGGTGCAACTCCACATCCACTTGGGTCACGTATGGAACGTGTACCCAGAGTGGGGAACGGAGACGGGAGAAGCGCTCAGTGGCACTGGCACCACATTCAGGAATGGGATTGGAAACCAGGACAGAGACGTGGATGTCATCGGAGGGGGCATGGAAACATGCCCAGAGAGTGGAGAGCTGGATACTTAGAAATCCAGGATTTGCACTCTTGGCAGGATTTATGGCTTACATGATTGGACAGACAGGAATTCAACGAACAGTCTTCTTTGTCCTCATGATGTTGGTCGCTCCATCCTATGGAATGCGATGCGTGGGAGTGGGGAATAGAGATTTTGTGGAAGGAGTCTCAGGAGGAACATGGGTCGACCTGGTGCTGGAACACGGAGGATGTGTTACAACCATGGCACAGGGAAAGCCAACCTTGGATTTTGAATTGATCAAGACAACAGCAAAGGAGGTAGCTCTATTAAGAACTTATTGCATAGAGGCCTCAATATCAAACATAACCACGGCAACAAGATGTCCAACACAAGGAGAACCTTATCTTAAAGAGGAACAAGACCAGCAGTACATTTGCAGAAGAGACGTGGTAGACAGAGGATGGGGTAATGGCTGTGGCCTATTTGGAAAAGGAGGAGTTGTAACATGCGCAAAGTTTTCATGCTCGGGGAAAATAACAGGCAACCTGGTCCAAGTTGAAAACCTTGAATACACAGTGGTTGTGACAGTTCATAATGGGGATGCCCACGCAGTGGGAAACAGCACGTCCAATCATGGAGTAACAACCACAATAACCCCCAGGTCACCATCGGTAGAAGTTAAACTACCAGATTATGGGGAACTGACACTCGATTGCGAACCCAGGTCCGGAATCGACTTTAACGAAATGATCCTGATGAAAATGAAAGGAAAAACATGGCTTGTGCACAAACAATGGTTCTTAGATCTACCCCTGCCATGGACAGCAGGAGCTGACACATCAGAAGTCCATTGGAATTATAAAGAGAGAATGGTGACGTTCAAAGTACCTCATGCCAAGAGACAGGATGTCACAGTGCTAGGATCCCAGGAAGGAGCCATGCACTCTGCCCTCACTGGAGCTACGGAGGTGGATTCTGGTGACGGAAACCACATGTTTGCAGGGCACCTAAAGTGCAAAGTGCGCATGGAAAAATTGAGGATCAAGGGAATGTCATACACGATGTGCTCAGGAAAGTTTTCCATCGACAAGGAAATGGCAGAAACGCAGCACGGGACAACAGTGGTGAAGGTCAAGTATGAAGGCACTGGGGCTCCATGCAAAATTCCAATAGAAATAAAAGACATGAATAAGGAAAAAGTGGTTGGACGCATTATTTCATCTATTCCCTTTGCTGAAAACACCAACAGCATAACCAATATTGAACTCGAACCCCCCTTTGGGGACAGCTACATAGTGATAGGCGCTGGAGACAGTGCATTGACACTCCATTGGTTTAGGAAGGGAAGTTCTATCGGGAAGATGTTTGAGTCCACTTATAGAGGTGCAAAAAGAATGGCCATTTTGGGTGAAACAGCATGGGATTTTGGCTCCGTTGGTGGATTGTTTACATCATTAGGGAAAGCTGTGCATCAGGTTTTTGGCAGTGTCTACACAACAATGTTTGGGGGAGTCTCATGGATGATCAGAATTCTCATTGGGATTTTAGTATTGTGGATCGGCACGAACTCAAGAAACACTTCAATGGCAATGTCATGCATAGCTGTTGGAGGAATCACCTTATTTCTTGGTTTTACGGTCCAAGCA41. DEN-4 prME wild type amino acid sequence (SEQ ID NO: 41)MIVAKHERGRPLLFKTTEGINRCTLIAMDVGEMCEDTVTYKCPLLVNTEPEDIDCWCNSTSTWVTYGTCTQSGERRREKRSVALAPHSGMGLETRTETWMSSEGAWKHAQRVESWILRNPGFALLAGFMAYMIGQTGIQRTVFFVLMMLVAPSYGMRCVGVGNRDFVEGVSGGTWVDLVLEHGGCVTTMAQGKPTLDFELIKTTAKEVALLRTYCIEASISNITTATRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGGVVTCAKFSCSGKITGNLVQVENLEYTVVVTVHNGDAHAVGNSTSNHGVTTTITPRSPSVEVKLPDYGELTLDCEPRSGIDFNEMILMKMKGKTWLVHKQWFLDLPLPWTAGADTSEVEIWNYKERMVTFKVPHAKRQDVTVLGSQEGAMHSALTGATEVDSGDGNHMFAGHLKCKVRMEKLRIKGMSYTMCSGKFSIDKEMAETQHGTTVVKVKYEGTGAPCKIPIEIKDMNKEKVVGRIISSIPFAENTNSITNIELEPPFGDSYIVIGAGDSALTLHWFRKGSSIGKMFESTYRGAKRMAILGETAWDFGSVGGLFTSLGKAVHQVFGSVYTTMFGGVSWMIRILIGILVLWIGTNSRNTSMAMSCIAVGGITLFLGFTVQA42. TVXDO24 (Capsid of DENV-2 and prME of DENV-4)Codon Optimized nucleotide sequence with mutations (SEQ ID NO: 42)ATGAACAACCAGCGCAAGAAGGCCAAAAACACTCCGTTCAATATGCTCAAGAGAGAGCGCAATCGGGTTTCTACGGTACAGCAGCTGACGAAGAGATTCTCCCTGGGCATGCTGCAAGGTCGCGGACCACTGAAGCTGTTCATGGCCCTTGTTGCATTTCTTAGGTTTCTTACAATTCCCCCCACTGCTGGAATCCTGAAGCGGTGGGGCACCATCAAAAAGTCCAAGGCTATTAATGTCCTCAGGGGGTTCAGGAAAGAGATTGGGCGGATGCTGAACATCCTTAATAGACGCAGACGGTCCGCTGGCATGATAATCATGCTGATCCCAACCGTCATGGCCTTTCATCTCAGCTCCCGCGATGGAGAACCTTTGATGATAGTCGCAAAACACGAACGGGGCAGGCCACTGCTTTTCAAGACTACTGAAGGCATCAACCGCTGCACCCTGATCGCAATGGACGTGGGTGAGATGTGCGAGGATACCGTGACTTATAAGTGCCCACTTCTCGTAAACACAGAGCCAGAAGACATTGATTGTTGGTGCAATTCTACCTCTACCTGGGTAACCTATGGAACTTGCACACAAAGCGGAGAAAGGAGAAGAGCCAAGCGGAGCGTTGCTCTGGCACCGCATTCCGGAATGGGACTTGAAACTAGAACAGAAACTTGGATGAGTAGCGAAGGAGCCTGGAAACATGCCCAACGGGTGGAAAGCTGGATTCTGCGCAACCCTGGATTCGCACTGCTTGCCGGTTTTATGGCATACATGATTGGACAGACCGGAATCCAGAGAACCGTTTTCTTTGTACTGATGATGCTGGTGGCTCCCTCTTATGGAATGCGATGTGTCGGCGTGGGCAATCGAGATTTTGTGGAAGGGGTCAGCGGGGGCACTTGGGTGGACCTCGTGCTGGAGCATGGAGGATGCGTTACAACCATGGCCCAAGGAAAACCTACACTTGATTTTGAACTGATAAAGACAACAGCTAAGGAAGTAGCCCTGTTGCGCACCTACTGTATCGAAGCTAGTATCTCTAACATCACTACAGCAACACGGTGCCCAACTCAGGGAGAACCCTATTTGAAGGAGGAGCAAGATCAGCAGTATATCTGTCGCCGAGATGTCGTGGACCGAGGATGGGGGAACGGCTGCGGGCTTTTTGGAAAAGGAGGCGTCGTGACCTGTGCTAAATTCAGTTGTTCAGGAAAGATTACGGGGAACCTCGTGCAGGTGGAGAACCTGGAATACACGGTGGTAGTAACAGTTCATAATGGGGACGCACACGCCGTAGGAAATAGCACCTCCAACCACGGCGTTACCACTACAATTACACCTAGAAGCCCTTCCGTGGAAGTTAAGCTGCCTGATTATGGGGAGCTCACCCTTGATTGCGAGCCCAGAAGTGGCATTGACTTTAACGAAATGATACTCATGAAGATGAAAGGAAAAACCTGGCTGGTACATAAACAGTGGTTCCTCGACCTTCCGCTCCCATGGACAGCAGGAGCCGACACCTCCGAGGTTCATTGGAATTACAAAGAGAGAATGGTTACTTTCAAGGTGCCACATGCGAAGCGCCAGGATGTGACAGTACTGGGATCCCAAGAAGGCGCCATGCACTCTGCCCTGACAGGCGCTACTGAGGTGGACTCCGGCGATGGAAATCACATGTTCGCGGGCCATCTGAAGTGTAAAGTAAGGATGGAGAAGCTGCGAATCAAAGGAATGTCCTATACGATGTGTTCAGGTAAGTTTTCTATTGACAAAGAAATGGCAGAAACCCAACATGGTACTACTGTGGTGAAGGTGAAATATGAAGGAACTGGAGCTCCATGTAAAATACCGATCGAGATCAAAGACATGAATAAGGAGAAAGTTGTGGGAAGAATCATAAGCAGCATTCCTTTTGCTGAGAATACTAACTCTATCACAAATATAGAACTTGAACCTCCGTTCGGTGATTCCTACATAGTAATCGGAGCCGGCGATTCAGCACTTACTCTGCACTGGTTCAGAAAAGGAAGTTCACTCGGAAAGGCTTTTGAGTCAACATATAGGGGCGCAAAGAGACTTGCAATTCTTGGGGAAACAGCTTGGGATTTCGGGAGCGTCGGTGGTCTGTTTACTTCCCTTGGAAAGGCGGTTCATCAAGTGTTTGGCTCAGTATACACCACAATGTTTGGGGGAGTGAGTTGGATGATCCGCATTCTTATCGGTATACTTGTGCTGTGGATTGGAACAAATTCAAGAAATACCAGTATGGCAATGTCATGTATTGCTGTGGGGGGGATAACTTTGTTTCTCG GGTTTACCGTGCAGGCATAG43. TVXDO24 amino acid sequence (SEQ ID NO: 43)MNNQRKKAKNTPFNMLKRERNRVSTVQQLTKRFSLGMLQGRGPLKLFMALVAFLRFLTIPPTAGILKRWGTIKKSKAINVLRGFRKEIGRMLNILNRRRRSAGMIIMLIPTVMAFHLSSRDGEPLMIVAKHERGRPLLFKTTEGINRCTLIAMDVGEMCEDTVTYKCPLLVNTEPEDIDCWCNSTSTWVTYGTCTQSGERRRAKRSVALAPHSGMGLETRTETWMSSEGAWKHAQRVESWILRNPGFALLAGFMAYMIGQTGIQRTVFFVLMMLVAPSYGMRCVGVGNRDFVEGVSGGTWVDLVLEHGGCVTTMAQGKPTLDFELIKTTAKEVALLRTYCIEASISNITTATRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGGVVTCAKFSCSGKITGNLVQVENLEYTVVVTVHNGDAHAVGNSTSNHGVTTTITPRSPSVEVKLPDYGELTLDCEPRSGIDFNEMILMKMKGKTWLVHKQWFLDLPLPWTAGADTSEVHWNYKERMVTFKVPHAKRQDVTVLGSQEGAMHSALTGATEVDSGDGNHMFAGHLKCKVRMEKLRIKGMSYTMCSGKFSIDKEMAETQHGTTVVKVKYEGTGAPCKIPIEIKDMNKEKVVGRIISSIPFAENTNSITNIELEPPFGDSYIVIGAGDSALTLHWFRKGSSLGKAFESTYRGAKRLAILGETAWDFGSVGGLFTSLGKAVHQVFGSVYTTMFGGVSWMIRILIGILVLWIGTNSRNTSMAMSCIAVGGI TLFLGFTVQA ZIKA44. ZIKV NS2B/NS3Pro wild-type nucleotide sequence (SEQ ID NO: 44)AGCTGGCCCCCTAGCGAAGTACTCACAGCTGTTGGCCTGATATGCGCATTGGCTGGAGGGTTCGCCAAGGCAGATATAGAGATGGCTGGGCCCATGGCCGCGGTCGGTCTGCTAATTGTCAGTTACGTGGTCTCAGGAAAGAGTGTGGACATGTACATTGAAAGAGCAGGTGACATCACATGGGAAAAAGATGCGGAAGTCACTGGAAACAGTCCCCGGCTCGATGTGGCGCTAGATGAGAGTGGTGATTTCTCCCTGGTGGAGGATGACGGTCCCCCCATGAGAGAGATCATACTCAAGGTGGTCCTGATGACCATCTGTGGCATGAACCCAATAGCCATACCCTTTGCAGCTGGAGCGTGGTACGTATACGTGAAGACTGGAAAAAGGAGTGGTGCTCTATGGGATGTGCCTGCTCCCAAGGAAGTAAAAAAGGGGGAGACCACAGATGGAGTGTACAGAGTAATGACTCGTAGACTGCTAGGTTCAACACAAGTTGGAGTGGGAGTTATGCAAGAGGGGGTCTTTCACACTATGTGGCACGTCACAAAAGGATCCGCGCTGAGAAGCGGTGAAGGGAGACTTGATCCATACTGGGGAGATGTCAAGCAGGATCTGGTGTCATACTGTGGTCCATGGAAGCTAGATGCCGCCTGGGACGGGCACAGCGAGGTGCAGCTCTTGGCCGTGCCCCCCGGAGAGAGAGCGAGGAACATCCAGACTCTGCCCGGAATATTTAAGACAAAGGATGGGGACATTGGAGCGGTTGCGCTGGATTACCCAGCAGGAACTTCAGGATCTCCAATCCTAGACAAGTGTGGGAGAGTGATAGGACTTTATGGCAATGGGGTCGTGATCAAAAATGGGAGTTATGTTAGTGCCATCACCCAAGGGAGGAGGGAGGAAGAGACTCCTGTTGAGTGCTTCGAG45. ZIKV NS3 Helicase wild type nucleotide sequence (SEQ ID NO: 45)CCTTCGATGCTGAAGAAGAAGCAGCTAACTGTCTTAGACTTGCATCCTGGAGCTGGGAAAACCAGGAGAGTTCTTCCTGAAATAGTCCGTGAAGCCATAAAAACAAGACTCCGTACTGTGATCTTAGCTCCAACCAGGGTTGTCGCTGCTGAAATGGAGGAAGCCCTTAGAGGGCTTCCAGTGCGTTATATGACAACAGCAGTCAATGTCACCCACTCTGGAACAGAAATCGTCGACTTAATGTGCCATGCCACCTTCACTTCACGTCTACTACAGCCAATCAGAGTCCCCAACTATAATCTGTATATTATGGATGAGGCCCACTTCACAGATCCCTCAAGTATAGCAGCAAGAGGATACATTTCAACAAGGGTTGAGATGGGCGAGGCGGCTGCCATCTTCATGACCGCCACGCCACCAGGAACCCGTGACGCATTTCCGGACTCCAACTCACCAATTATGGACACCGAAGTGGAAGTCCCAGAGAGAGCCTGGAGCTCAGGCTTTGATTGGGTGACGGATCATTCTGGAAAAACAGTTTGGTTTGTTCCAAGCGTGAGGAACGGCAATGAGATCGCAGCTTGTCTGACAAAGGCTGGAAAACGGGTCATACAGCTCAGCAGAAAGACTTTTGAGACAGAGTTCCAGAAAACAAAACATCAAGAGTGGGACTTTGTCGTGACAACTGACATTTCAGAGATGGGCGCCAACTTTAAAGCTGACCGTGTCATAGATTCCAGGAGATGCCTAAAGCCGGTCATACTTGATGGCGAGAGAGTCATTCTGGCTGGACCCATGCCTGTCACACATGCCAGCGCTGCCCAGAGGAGGGGGCGCATAGGCAGGAATCCCAACAAACCTGGAGATGAGTATCTGTATGGAGGTGGGTGCGCAGAGACTGACGAAGACCATGCACACTGGCTTGAAGCAAGAATGCTCCTTGACAATATTTACCTCCAAGATGGCCTCATAGCCTCGCTCTATCGACCTGAGGCCGACAAAGTAGCAGCCATTGAGGGAGAGTTCAAGCTTAGGACGGAGCAAAGGAAGACCTTTGTGGAACTCATGAAAAGAGGAGATCTTCCTGTTTGGCTGGCCTATCAGGTTGCATCTGCCGGAATAACCTACACAGATAGAAGATGGTGCTTTGATGGCACGACCAACAACACCATAATGGAAGACAGTGTGCCGGCAGAGGTGTGGACCAGACACGGAGAGAAAAGAGTGCTCAAACCGAGGTGGATGGACGCCAGAGTTTGTTCAGATCATGCGGCCCTGAAGTCATTCAAGGAGTTTGCCG CTGGGAAAAGA46. ZIKV N53 Helicase wild type amino acid sequence (SEQ ID NO: 46)PSMLKKKQLTVLDLHPGAGKTRRVLPEIVREAIKTRLRTVILAPTRVVAAEMEEALRGLPVRYMTTAVNVTHSGTEIVDLMCHATFTSRLLQPIRVPNYNLYIMDEAHFTDPSSIAARGYISTRVEMGEAAAIFMTATPPGTRDAFPDSNSPIMDTEVEVPERAWSSGFDWVTDHSGKTVWFVPSVRNGNEIAACLTKAGKRVIQLSRKTFETEFQKTKHQEWDFVVTTDISEMGANFKADRVIDSRRCLKPVILDGERVILAGPMPVTHASAAQRRGRIGRNPNKPGDEYLYGGGCAETDEDHAHWLEARMLLDNIYLQDGLIASLYRPEADKVAAIEGEFKLRTEQRKTFVELMKRGDLPVWLAYQVASAGITYTDRRWCFDGTTNNTIMEDSVPAEVWTRHGEKRVLKPRWMDARVCSDHAALKSFKEFAAGKR47. DEN-2 E protein wild-type amino acid sequence (SEQ ID NO: 47)MRCIGMSNRDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEAKQPATLRKYCIEAKLTNTTTESRCPTQGEPSLNEEQDKRFVCKHSMVDRGWGNGCGLFGKGGIVTCAMFRCKKNMEGKVVQPENLEYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSITEAELTGYGTVTMECSPRTGLDFNEMVLLQMENKAWLVHRQWFLDLPLPWLPGADTQGSNWIQKETLVTFKNPHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFKVVKEIAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLNWFKKGSSIGQMFETTMRGAKRMAILGDTAWDFGSLGGVFTSIGKALHQVFGAIYGAAFSGVSWTMKILIGVIITWIGMNSRSTSLSVTLVLVGIVTLYLGVMVQA

What is claimed is:
 1. A virus-like particle (VLP) comprising at leastone flavivirus structural protein; and at least one non-structuralflavivirus protein.
 2. The VLP of claim 1, wherein the structuralproteins comprise one or more of CPrME.
 3. The VLP of claim 1 or claim2, wherein the structural proteins consist of CPrME, PrME, CME, CPrE orME.
 4. The VLP of any of claims 1 to 3, wherein the non-structuralproteins comprise NS2B and/or NS3 proteins.
 5. The VLP of any of claims1 to 4, wherein the NS2B and/or NS3 proteins are truncated as comparedto wild-type.
 6. The VLP of any of claims 1 to 4, wherein the flavivirusis Dengue, Zika, yellow fever, Japanese encephalitis and/or West Nilevirus.
 7. The VLP of any of claims 1 to 6, wherein the VLP is a singlebivalent VLP that displays on its surface the E antigen of twoflavivirus virus serotypes or clades or a multivalent VLP that displayson its surface the E antigen of multiple flavivirus serotypes or clades.8. The VLP of claim 7, wherein the VLP comprises E antigens from atleast two different flaviviruses.
 9. A DNA construct comprisingsequences encoding flavivirus viral proteins used to assemble the VLP ofany of claims 1 to 8, the DNA construct comprising sequences encodingthe structural and non-structural proteins.
 10. The DNA construct ofclaim 9, further comprising one or more sequences encoding a linkerbetween one or more of the sequences encoding the structural andnon-structural proteins.
 11. The DNA construct of claim 10, wherein thelinker comprises amino acids corresponding to amino acids 1 to 8 or 9 or10 of NS1 and amino acids corresponding to 186 or 187 or 188 or 189 toamino acids corresponding to 218 of NS2A; amino acid 1 to 8 or 9 or 10of NS1, amino acids 1 to 24 or 25 or 26 or 27 or 28 or 29 or 30 or 31 or32 of NS2A, amino acids 186 or 187 or 188 or 189 to 218 of NS2A; aminoacids 190 or 191 or 192 or 193 to amino acids corresponding to 225 ofNS2A; amino acids 190 or 191 or 192 or 193 to amino acids 225 of NS2A;amino acids 1 to 8 or 9 or 10 of NS1 and the second transmembrane domainof NS2B; amino acid 1 to 8 or 9 or 10 of NS1 and the first transmembranedomain of NS2A; and amino acid 1 to 8 or 9 or 10 of NS1 and the Cterminal portion of NS2B comprising the second transmembrane domain tothe end of the protein.
 12. The DNA construct any of claims 9 to 11,wherein the non-structural protein optionally includes a full lengthNS2B and a full NS3 operably linked directly to the structural proteinsCprME.
 13. The DNA construct any of claims 9 to 11, wherein the NS3protease active site is modified such that its enzymatic activity isenhanced.
 14. The DNA construct of any of claims 9 to 11, wherein thefurin protease cleavage site between pr and M protein is modified bysubstituting amino acids residues at position P3 with hydrophobic onesuch that furin cleavage is enhanced.
 15. The DNA construct of any ofclaims 9 to 11, wherein the sequence encoding the E protein is modifiedto enhance VLP assemble and release.
 16. A method of producing a VLP,the method comprising introducing into a host cell one or more DNAconstructs according to any of claims 9 to 14 under conditions such thatthe cell produces the VLP.
 17. The method of claim 16, wherein the hostcell is a eukaryotic cell selected from the group consisting ofmammalian, yeast, insect, plant, amphibian and avian cells.
 18. Themethod of claim 16 or claim 17, wherein the cells are cultured attemperatures ranging from 25° C. to 37° C.
 19. A VLP generated by themethod of any of claims 16 to
 18. 20. An immunogenic compositioncomprising at least one VLP according to any of claims 1 to 8 or
 19. 21.The immunogenic composition of claim 20, further comprising an adjuvant.22. The immunogenic composition of claim 20 or claim 21, wherein thecomposition comprises at least two VLPs comprising different flavivirusE proteins.
 23. A method of generating an immune response to one or moreflaviviruses in a subject, the method comprising administering to thesubject an effective amount of the immunogenic composition according toany of claims 20 to
 22. 24. The method of claim 23, wherein thecomposition is administered mucosally, intradermally, subcutaneously,intramuscularly, or orally.
 25. The method of claim 23 or claim 24,wherein the immune response vaccinates the subject against multipleserotypes or clades of one or more flaviviruses.
 26. The method of anyof claims 23 to 25, wherein the subject is a human.